Imaging and treatment of pathophysiologic conditions by cerenkov radiation

ABSTRACT

The present disclosure discloses methods and compositions for A administering Cerenkov radiation-induced therapy (GRIT). In an aspect, the invention encompasses a composition comprising at least two radiation-sensitive molecules. In another aspect, the invention encompasses a composition comprising a radiation-sensitive molecule and a targeting agent. In still another aspect, the invention encompasses a method for administering Gerenkov radiation-induced therapy (GRIT) to a target tissue in a subject. The method comprises administering to the subject an effective amount of a composition B comprising at least one radiation-sensitive molecule and administering to the subject an amount of a Gerenkov radiation (GR)-emitting radionuclide effective to activate the radiation-sensitive molecule, thereby administering GRIT to the target tissue in the subject.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of PCT ApplicationPCT/US2015/014095, filed Feb. 2, 2015, which claims the benefit of U.S.provisional application No. 61/934,073, filed Jan. 31, 2014, and U.S.provisional application No. 62/012,086, filed Jun. 13, 2014, each of thedisclosures of which is hereby incorporated by reference in itsentirety.

GOVERNMENTAL RIGHTS

This invention was made with government support under R01 EB008111, NCIR01 CA171651 and SIG 1S10RR031625-01 awarded by the NIH and under CCF0963742 awarded by the NSF. The government has certain rights in theinvention.

FIELD OF THE INVENTION

The present disclosure discloses methods and compositions foradministering Cerenkov radiation-induced therapy (CRIT).

BACKGROUND OF THE INVENTION

Photodynamic Therapy (PDT) is a therapeutic procedure to destroy tissue,preferably pathological tissue, for example, cancer tissue or tissue inblood vessels that occur in disorders characterized byhypervascularization or proliferation of neovascular networks. Incancer, PDT can be locally administered as a primary therapy for earlystage disease, palliation of late stage disease, or as a surgicaladjuvant for tumors that show loco-regional spread.

In PDT, a photosensitizing agent (termed a “photosensitizer”) isdelivered to the target tissue and then radiation, most usually light ofwavelengths between 250-1000 nm is applied to the target tissue. Thus,photosensitizing agents are activated by electromagnetic (EM) radiation.This activation results in the photochemical transfer of the energy bythe photosensitizer-molecules to a variety of other molecules in thetissue, resulting in the generation of reactive radical speciesincluding, amongst others, singlet oxygen, the superoxide radical, andperoxides and peroxide radicals. The activation of the photosensitizingagent in the tissue leads to, amongst other processes, the generation ofradicals and, ultimately, the destruction of the target tissue, or theinitiation of biological processes that result in the desired effectupon the target tissue.

However, the limited penetrability of light in tissues remains a largelimiting factor in the use of PDT for the treatment of cancer,specifically cancers located within deeper tissue. Therefore, there is aneed for methods of PDT, and phototherapy in general, that improve thetissue depth of penetration producing clinical benefits in deep tumors.Furthermore, there is a need to reduce the level of radiation needed todecrease the toxicity associated with POT.

SUMMARY OF THE INVENTION

In an aspect, the invention encompasses a composition comprising atleast two radiation-sensitive molecules.

In another aspect, the invention encompasses a composition comprising aradiation-sensitive molecule and a targeting agent.

In still another aspect, the invention encompasses a method foradministering Cerenkov radiation-induced therapy (CRIT) to a targettissue in a subject. The method comprises administering to the subjectan effective amount of a composition comprising at least oneradiation-sensitive molecule and administering to the subject an amountof a Cerenkov radiation (CR)-emitting radionuclide effective to activatethe radiation-sensitive molecule, thereby administering CRIT to thetarget tissue in the subject.

In still another aspect, the invention encompasses a method of detectinga tumor in a subject. The method comprises administering to the subjectan effective amount of a composition comprising at least oneradiation-sensitive molecule, administering to the subject an amount ofa Cerenkov radiation (CR)-emitting radionuclide effective to activatethe radiation-sensitive molecule, and imaging the subject for a signalcorresponding to the radiation-sensitive molecule, wherein a signalcorresponding to the radiation-sensitive molecule indicates detection ofthe tumor.

In still yet another aspect, the invention encompasses a method fortreating a tumor in a subject. The method comprises administering to thesubject an effective amount of a composition comprising at least oneradiation-sensitive molecule; and an amount of a Cerenkov radiation(CR)-emitting radionuclide effective to activate the radiation-sensitivemolecule, thereby treating the tumor.

BRIEF DESCRIPTION OF THE FIGURES

The application file contains at least one drawing executed in color.Copies of this patent application publication with color drawing(s) willbe provided by the Office upon request and payment of the necessary fee.

FIG. 1A, FIG. 1B, FIG. 1C, FIG. 1E, FIG. 1F, FIG. 1G, FIG. 1H and FIG.1I depict illustrations, images and graphs of coated photocatalytic TiO₂as photosensitizers for CR-PDT. (FIG. 1A-FIG. 1C) Schematic of (FIG. 1A)TiO₂ nanoparticles, (FIG. 1B) TiO₂ nanoparticles coated with PEG (MW:400 Da) and (FIG. 1C) TiO₂ nanoparticles coated with dextran (MW: 5,000Da) moieties (not to scale). (FIG. 1D-FIG. 1F) Transmission electronmicroscopy images of TiO₂ aggregates (FIG. 1D; scale bar, 400 nm),TiO₂-PEG (FIG. 1E; scale bar, 100 nm) and TiO₂-dextran (FIG. 1F; scalebar, 100 nm). (FIG. 1G-FIG. 1I) Dynamic light scattering intensity plotshowing the distribution of hydrodynamic diameter of TiO₂ (FIG. 1G),TiO₂-PEG (FIG. 1H) and TiO₂-dextran (FIG. 1I).

FIG. 2A, FIG. 2B, FIG. 2C, FIG. 2D, FIG. 2E and FIG. 2F depict graphs,images and an illustration of in vitro CR-PDT using TiO₂ and ⁶⁴Cu. (FIG.2A) Comparison of cytotoxicity of TiO₂, TiO₂-dextran and TiO₂-PEG onBxPC-3 cells after incubation with 0.5 mCi of ⁶⁴Cu for 72 h. Values aremeans±s.e.m. (experiments for each group were run in triplicates). (FIG.2B) In vitro CR-PDT comparing the cytotoxicity of 0.1, 0.25, 0.5, 1 mCiof ⁶⁴Cu on BxPC-3 cells loaded with 2.5 μg/ml TiO₂-PEG using MTS assayafter 72 h. Values are means±s.e.m. (experiments for each group were runin triplicates). (FIG. 2C) Plot showing the relative change in hydroxyland superoxide radicals generated by BxPC-3 cells with 2.5 μg/mlTiO₂-PEG and 0.05, 0.1, 0.25, 0.5, 1 mCi of ⁶⁴Cu, using HPF and Mitosoxdye, respectively. Values are means±s.e.m. (experiments for each groupwere run in triplicates). (FIG. 2D, FIG. 2E) Confocal microscopy imageof merged bright-field and fluorescence images of Matrigel™ suspendedBxPC-3 cells with extracellular TiO₂-PEG (FIG. 2D) and intracellularTiO₂-PEG (FIG. 2E), with 0.25 mCi ⁶⁴Cu. Live/Dead® cell viability stainwas used to distinguish live cells (green) from dead cells (red). Scalebar, 100 μm. (FIG. 2F) Schematic of ⁶⁴Cu generated CR mediating PDT oninternalized TiO₂ (not to scale).

FIG. 3A, FIG. 3B, FIG. 3C, FIG. 3D, FIG. 3E and FIG. 3F depict graphsand images of in cellulo fluorescence imaging of TiO₂. (FIG. 3A)Absorption spectrum of TiO₂. (FIG. 3B) Fluorescence spectrum of TiO₂ andtumor cell internalized TiO₂, excited at 275 nm. CPS, counts per second.(FIG. 3C) Epifluorescence microscopy images of BxPC-3 cells loaded with60 μg/ml of TiO₂-PEG, taken using DAPI, FITC and Cy5 filters. Scale bar,50 μm. (FIG. 3D) Confocal microscopy images of BxPC-3 cells loaded with60 μg/ml of TiO₂-PEG. Scale bar, 100 μm (FIG. 3E) Magnified confocalimage of a single BxPC-3 cell showing the crystalline TiO₂ particles inthe cytoplasm. Scale bar, 15 μm. (FIG. 3F) A 3D slice of the same cellvisualized in the z-plane showing the uniform distribution of TiO₂ inthe cytoplasm.

FIG. 4A, FIG. 4B, FIG. 4C, FIG. 4D and FIG. 4E depict graphs and imagesof in vivo luminescence imaging of TiO₂. (FIG. 4A) Luminescence spectraof CR from ⁶⁴Cu. (FIG. 4B) Luminescence spectra of 60, 125, 250, 500μg/ml of TiO₂ admixed with 0.25 mCi of ⁶⁴Cu in vitro, recorded indifferent channels: GFP (515-575 nm), DsRed (575-650 nm), Cy5.5 (685-770nm), and ICG (810-875 nm). Values are means±s.e.m. (experiments for eachgroup were run in triplicates). (FIG. 4C) In vivo luminescence images ofsubcutaneous tumor mimics in Balb/c mice, created by mixing Matrigel™with different titrations of TiO₂ (60, 125, 250, 500 μg/ml) and 0.25 mCiof ⁶⁴Cu. (n=3 mice per group). Color legend bar is the same for c&d.(FIG. 4D) In vivo luminescence image of BxPC-3 tumor in Athymic nu/numice after injecting 250 μg/ml of TiO₂ and 0.25 mCi of ⁶⁴Cuintratumorally. (n=3 mice per group). (FIG. 4E) In vitro phantom studiescarried out with 0.1 mCi of ⁶⁴Cu, 0.1 mCi ⁶⁴Cu admixed with 1 mg/mlTiO₂, and 0.1 mCi of ^(99m)Tc admixed with 1 mg/ml TiO₂, in each well.

FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D, FIG. 5E, FIG. 5F, FIG. 5G, FIG. 5H,FIG. 5I, FIG. 5J, FIG. 5K and FIG. 5L depict graphs and images of invivo CR-PDT in pancreatic (BxPC-3) and fibrosarcoma (HT1080) solid tumorxenografts. (FIG. 5A) In vivo CR-PDT of tumor mimics: Growth ofMatrigel™ suspended seed culture of BxPC-3 cells implantedsubcutaneously in different groups of Athymic nu/nu mice. Values aremeans±s.e.m. (n=6 mice per group). (FIG. 5B) In vivo CR-PDT of solidtumors: Growth of BxPC-3 and HT1080 tumors in different groups ofAthymic nu/nu mice including appropriate controls. Values aremeans±s.e.m. (n=4 mice per group). Inset: Change in tumor size within 10d. (FIG. 5C) Representative photographs of BxPC-3 tumor bearing miceinjected with 2.5 μg/ml of TiO₂-PEG and 0.25 mCi of ⁶⁴Cu intratumorallyat day 0, 10 and 45. Scale bar, 5 mm. (FIG. 5D) Representativephotograph of HT1080 tumor bearing mice injected with 2.5 μg/ml ofTiO₂-PEG and 0.25 mCi of ⁶⁴Cu intratumorally at day 0, 3 and 45. Scalebar, 5 mm. Complete tumor elimination was achieved after PDT at day 45(dotted circle). (FIG. 5E) Untreated H&E stained BxPC-3 tumor sectionshowing a stromal architecture. Scale bar, 1 mm. (n=4 histologicalsections per group). (FIG. 5F) H&E stained BxPC-3 tumor section 60 dafter commencement of PDT, showing minimal change in the tumorarchitecture except the top right edge that could be perhaps associatedwith needle entry and injection site damage. Scale bar, 1 mm. (n=4histological sections per group). (FIG. 5G) Magnified epifluorescenceimage of the tumor section showing fluorescence from residual TiO₂particles entrapped in the stroma. Scale bar, 200 μm. (FIG. 5H)Magnified H&E stained section of normal BxPC-3 tumors showing densestroma surrounding islands of tumor cells. Scale bar, 200 μm. (FIG. 5I)H&E stained HT1080 tumor section before PDT showing typical herringbonearchitecture of fibrosarcoma. Scale bar, 1 mm. (n=4 histologicalsections per group). (FIG. 5J) H&E stained HT1080 tumor section 3 dafter commencement of PDT showing extensive necrotic centers anddestruction of the tumor architecture. Scale bar, 1 mm. (n=4histological sections per group). (FIG. 5K) Magnified epifluorescenceimage of the HT1080 tumor section showing localization and enrichment ofTiO₂ in the tumor architecture. Scale bar, 200 μm. (FIG. 5L) MagnifiedH&E stained section of normal HT1080 tumors. Notice the relative absenceof stroma and the arrangement of tumor cells. Scale bar, 200 μm.

FIG. 6 depicts a comparison of cytotoxicity of 3 μg/ml TiO₂ and itsadducts, TiO₂-PEG and TiO₂-dextran, using MTS assay.

FIG. 7A and FIG. 7B depict the effect of photosensitive particles andradionuclides on cell viability. (FIG. 7A) MTS cytotoxicity assayquantifying concentration of Tc-Tf, TiO₂-Tf and TiO₂-PEG effecting cellviability. The control group was considered to be 100% viable. (FIG. 7B)MTS cytotoxicity assay quantifying concentration of ⁶⁴Cu and FDGeffecting cell viability. HT1080 cells were used and the values aremeans±s.e.m. (experiments for each group were run in triplicates).

FIG. 8 depicts a TEM image of HT1080 cell with TiO₂-Tf in theendo-lysosomal compartments (arrows). Scale bar, 2 μm.

FIG. 9 depicts confocal microscopy images of merged bright-field andfluorescence images comparing the degree of necrotic cell death causedby 0.1, 0.25, 0.5, 1 mCi/100 μl of 64Cu on tumor cells with 2.5 μg/mlTiO₂-PEG after 72 h. PI dye was used to stain nuclei as a measure ofcell viability. Majority of cells incubated with >0.1 mCi/100 μl of ⁶⁴Custained positive with PI. Scale bar, 20 μm.

FIG. 10 depicts confocal images of merged bright-field and fluorescenceimages comparing the degree of hydroxyl radical generation caused by0.1, 0.25, 0.5, 1 mCi/100 μl of ⁶⁴Cu on tumor cells with 2.5 μg/mlTiO₂-PEG after 4 h. HPF dye was used to stain the cells. The greenfluorescence from cells depicts increased hydroxyl radical generation.Highest fluorescence intensity was recorded from cells incubated with0.25 mCi/100 μl of ⁶⁴Cu. Scale bar, 20 μm.

FIG. 11 depicts epilfluorescence images of BxPC-3 cells withinternalized TiO₂-PEG at various concentrations with an exposure time of450 ms and 4×4 binning using Cy5 filter with an excitation and emissionwavelength of 630 nm and 700 nm, respectively. Scale bars: 150 μm.

FIG. 12A and FIG. 12B depict the fluorescent spectrum of TiO₂ particles.(FIG. 12A) Fluorescence spectrum of TiO₂ with excitation at 488 nm.(FIG. 12B) Fluorescence spectrum of TiO₂ with excitation at 633 nm.

FIG. 13A, FIG. 13B and FIG. 13C depict titanium dioxide and titanocenephotoagents for CRIT. (FIG. 13A) Schematic of CR mediated excitation ofTiO₂ nanoparticles to generate cytotoxic hydroxyl and superoxideradicals from water and dissolved oxygen, respectively, throughelectron-hole pair generation. CR is generated by PET radionuclides (notto scale). (FIG. 13B) Schematic of CR mediated excitation of Tc togenerate a cyclopentadienyl radical and a titanium-centered radicalthrough photofragmentation (not to scale). In aerated media, theradicals transform into more potent peroxyl radicals. (FIG. 13C)Schematic illustrating the development of TiO₂-PEG,TiO₂-Tf by coatingTiO₂ with Tf and subsequent generation of TiO₂-Tf-Tc construct by simpleaddition of Tc, which docks into the iron binding site of Tf (not toscale). Below (left to right) are the Transmission electron microscopyimages of TiO₂-PEG, TiO₂ aggregates, TiO₂-Tf and TiO₂-Tf-Tc (right).Scale bar, 50 nm.

FIG. 14A, FIG. 14B, FIG. 14C, FIG. 14D, FIG. 14E, FIG. 14F, FIG. 14G,FIG. 14H, FIG. 14I, FIG. 14J, FIG. 14K and FIG. 14L depict cellularuptake of photoagents and in vitro CRIT assessment. (FIG. 14A) Electronmicroscope image of a HT1080 tumor cell showing internalized andendo-lysosomal localization of the TiO₂-Tf constructs (arrows). Scalebar, 2 μm. Inset shows two lysosomal compartments with TiO₂-Tf. Scalebar, 400 nm. (FIG. 14B) Cell viability assay comparing the TiO₂-Tf,Tc-Tf and TiO₂-Tf-Tc constructs with and without exposure to ⁶⁴Cu andFDG on HT1080 cells. Values are means±s.e.m. (experiments for each groupwere run in triplicates). ***P<0.001. (FIG. 14C) Examples of alkalinecomet assay results. The images show undamaged and damaged DNA as aresult of free radical damage and apoptosis. Image marked (i) isrepresentative of undamaged DNA, from the controls, including untreatedcells and either exposed to NPS or radionuclide alone. Notice there isnegligible DNA in the tail (0.15%). In comparison, cells treated withthe NPS and radionuclide, show considerable DNA damage as shown in (ii,iii, iv). Cells in the same treatment group exhibited variable DNAdamage, such as 22.32%, 45.87% and 71.84% DNA in the tail. Thefluorescence intensity is represented in pseudocolor. (FIG. 14D) Cellsundergoing CRIT demonstrated an overall higher percent of damaged DNA.100 cells were counted from each group. Values are means±s.e.m.**P<0.01. (FIG. 14E) EM image of a normal HT1080 cell. Scale bar, 3 μm.(FIG. 14F) EM image showing a necrotic cell that was treated withTiO₂-Tf (arrows) and FDG. Notice loss of cell membrane integrity andhighly vacuolated cytoplasm. Scale bar, 2 μm. (FIG. 14G) EM imageshowing an apoptotic cell that was treated with TiO₂-Tf (arrows) andFDG. Notice surface blebbing and condensed chromatin. Scale bar, 1.4 μm.(FIG. 14H) EM image of an apoptotic cell that was treated with Tc-Tf andFDG. Notice nuclear fragmentation and chromatin margination. Scale bar,1.4 μm. (FIG. 14I) Confocal laser scanning microscopy images of HT1080cells comparing the difference in propidium iodide uptake betweenTiO₂-Tf and FDG treated cells and Tc-Tf and FDG treated cells. Themostly necrotic TiO₂-Tf treated cells show a high uptake of PI andclassical nuclear staining. The oncotic cells in Tc-Tf treated samplesshow light nuclear staining in comparison. Scale bar, 20 μm. (J) Thepercentage of cells which show positive PI staining of nuclei are muchlower in Tc-Tf and FDG treated cells. Values are means±s.e.m.(experiments for each group were run in triplicates). *P<0.05. (FIG.14K) Comparison between HT1080 cells not undergoing and undergoing CRITwith TiO₂-Tf and Tc-Tf show higher output of free radicals such ashydroxyl, superoxide and peroxyl species as measured using HPF andMitosox fluorescent dyes. Values are means±s.e.m. (experiments for eachgroup were run in triplicates). (FIG. 14L) Confocal microscopy image ofmerged bright-field and fluorescence images of Matrigel™ suspended cellswith extracellular TiO₂ (left) and intracellular TiO₂ (right), with 0.5mCi/0.1 ml ⁶⁴Cu. Live/Dead® cell viability stain was used to distinguishlive cells (green) from dead cells (red). Scale bar, 20 μm.

FIG. 15A, FIG. 15B, FIG. 15C and FIG. 15D depict CRIT throughintratumoral administration of TiO₂ and ⁶⁴Cu. (FIG. 15A) In vivo CRITthrough a one-time intratumoral administration of PEGylated TiO₂ and⁶⁴Cu in HT1080 tumor bearing Athymic nu/nu mice. Toxicity throughelemental Cu was eliminated by using non-radiactive CuCl₂, with andwithout TiO₂-PEG. Values are means±s.e.m. (n=4 mice per group). (FIG.15B) Representative photographs at day 1, 3 & 45 of HT1080 tumor bearingmice injected with a single dose of 2.5 μg/ml of TiO₂-PEG and 0.5mCi/0.1 ml of ⁶⁴Cu intratumorally at day 1. Scale bar, 5 mm. Completetumor elimination was achieved after PDT at day 45 (dotted circle).(FIG. 15C) H&E stained HT1080 tumor section before PDT showing typicalherringbone architecture of fibrosarcoma. Scale bar, 1 mm. (n=4histological sections per group). (FIG. 15D) H&E stained HT1080 tumorsection 3 d after commencement of PDT showing extensive necrotic centersand destruction of the tumor architecture. Scale bar, 1 mm. (n=4histological sections per group).

FIG. 16A, FIG. 16B, FIG. 16C, FIG. 16D, FIG. 16E, FIG. 16F and FIG. 16Gdepict in vivo biodistribution and CRIT through systemicallyadministered photoagents and FDG. (FIG. 16A) In vivo biodistribution ofTiO₂-Tf and Tf alone using Alexa 680 labeled holo-Tf in HT1080 tumorbearing Athymic nu/nu mice over a period of 24 h. Values aremeans±s.e.m. (n=5 mice per group). (FIG. 16B) In vivo CRIT through aone-time systemic administration of the constructs and FDG in HT1080tumor bearing Athymic nu/nu mice. Values are means±s.e.m. (n=6 mice pergroup). **P<0.01, ***P<0.001. (FIG. 16C) Kaplan-Meier survival curvesrepresenting treatment with 0.87 mCi/0.1 ml FDG. *** P<0.001. (FIG. 16D)Survival curves representing treatment with 0.14 and 0.43 mCi/0.1 ml FDG(n=4 mice per group). **P<0.01. (FIG. 16E) FDG-PET images of untreated(left) mouse with bilateral HT1080 tumors and after CRIT (30 d), imagedby administering 0.19 mCi/0.1 ml FDG i.v. Notice the right tumor inmouse undergoing CRIT displays a necrotic zone. (FIG. 16F) StandardUptake value of FDG is considerably low in mouse that underwent CRIT.***P<0.001. (FIG. 16G) Histological analysis of H&E stained HT1080 tumorsections from an untreated mouse are compared to mice that underwentCRIT. Normal tumor tissue is marked as T, necrotic tissue as N, anddenuded areas suggesting macrophage assisted clearance is marked as *.Magnified images show tumor infiltrating lymphocytes in the treatedtumor sections.

FIG. 17A, FIG. 17B, FIG. 17C, FIG. 17D and FIG. 17E depict spectralcharacterization of TiO₂ and Tc. (FIG. 17A) Absorption spectrum of TiO₂in water. (FIG. 17B) Absorption spectrum of Tc in water/DMSO (95/5%).(FIG. 17C) Emission spectrum of CR from ⁶⁴Cu. (FIG. 17D) Fluorescencespectrum of TiO₂, excited at 275 nm. CPS, counts per second. (FIG. 17E)In vitro luminescence studies carried out with 0.1 mCi/100 μl of ⁶⁴Cu,0.1 mCi/100 μl ⁶⁴Cu admixed with 1 mg/ml TiO₂, and 0.1 mCi/100 μl of^(99m)Tc admixed with 1 mg/ml TiO₂, in each well. Images were capturedin the GFP (515-575 nm) channel.

FIG. 18A and FIG. 18B depict cytoxicity and apoptosis observed withphotosensitizer nanoparticles versus gold nanoparticles. (FIG. 18A) MTScytotoxicity assay comparing viability of tumor cells incubated withTiO₂-Tf and gold nanoparticles along with FDG. No change in metabolicprofile and proliferation rate of cells incubated with goldnanoparticles was noticed. Values are means±s.e.m. **P<0.01. (FIG. 18B)Propidium iodide uptake assay comparing untreated, TiO₂-Tf and goldnanoparticles along with FDG. Minimal uptake of PI was observed,suggesting no damage to the cell membrane due to radiosensitization ofcells. Values are means±s.e.m. (experiments for each group were run intriplicates).

FIG. 19A, FIG. 19B, FIG. 19C and FIG. 19D depict the biodistribution ofTf and TiO₂-Tf. (FIG. 19A) In vivo biodistribution profile of Alexa 680labeled Tf in HT1080 tumor bearing Athymic nude mice at 24 h followingtail vein injection (n=5). (FIG. 19B) Ex vivo fluorescence image ofdissected organs from (A). Notice the high fluorescence from bloodsuggesting circulating Tf. (FIG. 19C) In vivo biodistribution profile ofAlexa 680 labeled TiO₂-Tf in HT1080 tumor bearing Athymic nude mice at24 h following tail vein injection (n=5). (FIG. 19D) Ex vivofluorescence image of dissected organs from (FIG. 19C). Fluorescenceimaging was performed using an excitation and emission wavelength of 685nm and 720 nm, respectively.

FIG. 20 depicts images of histological analysis of H&E stained liver andkidney sections before and after treatment are shown to demonstrate nosignificant lesions in these organs indicating absence of systemictoxicity due to CRIT.

FIG. 21 depicts a graph of biodistribution studies of TiO₂-Tf in mice(n=3) with HT1080 tumors at various timepoints.

FIG. 22 depicts a schematic of the treatment plan.

FIG. 23 depicts a graph of tumor growth curves during CR-PDT.

FIG. 24A, FIG. 24B, FIG. 24C and FIG. 24D depict graphs and imagesshowing the composition and phase characterization of TiO₂-Tf. (FIG.24A) EDX spectra of unprocessed TiO₂ with the peaks labelled as Ti fortitanium, 0 for oxygen and C for carbon. (FIG. 24B) EDX spectra ofTiO₂-Tf with a pronounced C peak suggesting presence of the protein Tfon the surface of TiO₂. (FIG. 24C) Electron diffraction of unprocessedTiO₂ with ring measurements matching the crystal pattern of anatase formof TiO₂, from diffraction file: 21-1272. (FIG. 24D) Electron diffractionpattern of TiO₂-Tf with ring measurements and crystal structureidentical to that of TiO₂.

FIG. 25A and FIG. 25B depict graphs showing the serum stability ofTiO₂-Tf NPS. (FIG. 25A) Comparison of fluorescence intensity betweenTiO₂-AlexaTf NPS incubated in foetal bovine serum for 24 h and untreatedsamples (ns: not significant). (FIG. 25B) Comparison of unlabelledTiO₂-Tf and unprocessed TiO₂ incubated with Alexa 680 labelled albumin.Values are means±s.e.m. (experiments for each group were run intriplicates). **P<0.01.

FIG. 26A and FIG. 26B depict images and a graph showing the cellularuptake of NPS. (FIG. 26A) In cellulo uptake of TiO₂-Tf labelled withAlexa 680 dye and successful blocking with holo-Tf suggesting Tfreceptor mediated internalization as the mechanism of uptake. Scale bar,20 μm. (FIG. 26B) Quantitation of successful blocking of TiO₂-Tfinternalization by saturating doses of holo-Tf in HT1080 cells. Valuesare means±s.e.m. (experiments for each group were run in triplicates andreplicated 2×). **P<0.01. Tf receptor mediates endocytosis of Tf-coatedNPS in tumour cells.

FIG. 27A, FIG. 27B and FIG. 27C depict images and a graph showing the Invivo blocking of TiO₂-Tf uptake by HT1080 tumours. (FIG. 27A) Organbiodistribution of TiO₂-AlexaTf. (FIG. 27B) Organ biodistribution ofTiO₂-AlexaTf after administration of holo-Tf to block Tf receptors.(FIG. 27C) Comparison of biodistribution of TiO₂-AlexaTf with andwithout blocking.

FIG. 28 depicts a lipid peroxidation assay using BODIPY 581/591 C11reagent on HT1080 cells showing a higher degree of lipid peroxidation incells treated with Tc and FDG. Values are means±s.e.m. *P<0.05, **P<0.01***P<0.001.

FIG. 29 depicts a graph showing loss of mitochondrial membrane potentialdue to CRIT. Mitochondrial membrane potential changes detected byMitotracker Green dye as a result of CRIT. Values are means±s.e.m.*P<0.05, **P<0.01.

FIG. 30A and FIG. 30B depict TEM analysis of tumor uptake of TiO₂-Tf-Tc.(FIG. 30A) TEM image of tumour sections showing localization of theTiO₂-Tf-Tc constructs (arrow) in tumour cells after i.v. administration.Scale bar, 500 nm. (FIG. 30B) TEM image of tumour sections of miceinjected with TiO₂-PEG showing absence of TiO₂ in the tumour cells.Scale bar, 1 μm. High tumour uptake and retention of Tf-coated NPSrelative to non-tumour tissues demonstrate the feasibility of CRIT viai.v. administration of CR source following selective retention of theNPS in tumours.

FIG. 31A, FIG. 31B, FIG. 31C and FIG. 31D depict TEM analysis of CRIT invivo. (FIG. 31A) TEM image of tumour section extracted from untreatedmice showing healthy cells. Scale bar, 3 μm (FIG. 31B) TEM image oftumour section extracted from mice that underwent CRIT showing majorityof cells are apoptotic. Scale bar, 3 μm. (FIG. 31C) Magnified TEM imageof (i) showing internalized NPS (arrows) in apoptotic tumour cells.Scale bar, 500 nm. (FIG. 31D) TEM image of tumour section from necroticregion showing necrotic cells with internalized NPS (arrows). Scale bar,2 μm.

FIG. 32 depicts a graph showing the change in murine weights inuntreated and treated groups of mice. TiO₂-PEG and chelated ⁶⁴Cu wereadministered intratumourally and monitored over 4 months.

FIG. 33 depicts a graph showing in vivo CRIT in A549 tumour bearingAthymic nu/nu mice using TiO₂-Tf-Tc and FDG. Values are means±s.e.m.(n=4 mice per group). Experiments were replicated 2×. ***P<0.001.

FIG. 34A, FIG. 34B, FIG. 34C, FIG. 34D and FIG. 34E depict graphs andimages showing in vivo CRIT in U266 multiple myeloma tumor model. (FIG.34A) In vivo CRIT in U266 Multiple myeloma tumor model grown in NSGmice. (FIG. 34B) Kaplan-Meier survival curves showing increase in mediansurvival by ˜ 8 d in targeted CRIT. (FIG. 34C) Serum proteinelectrophoresis analysis showing serum γ-globulin levels were lower intargeted CRIT. Ex vivo fluorescence images of (FIG. 34D) untreated and(FIG. 34E) CRIT-treated tumors using filter for GFP (Ex/Em: 488/535 nm).

FIG. 35A, FIG. 35B and FIG. 35C depict flow cytometry plots of multiplemyeloma cells. (FIG. 35A) Flow cytometry of MM1.S cell line using CD71antibodies showing high expression of TfR (99%). (FIG. 35B) Flowcytometry on T cells with CD71 and CD4 antibodies showing low expressionof TfR (2%). (FIG. 35C) Flow cytometry of B cells with CD71 and CD19antibodies showing low expression of TfR (25%).

FIG. 36 depicts MTS cell viability assay (48 h) demonstrates higherdegree of cell death when treated with TiO₂-Tf-Tc+FDG, in both MM celllines. 1. Untreated. 2. Positive control (staurosporine). 3. TiO₂-Tf-Tc.4. FDG 31 MBq/0.1 ml. 5. TiO₂-Tf-Tc+FDG.

FIG. 37 depicts diodistribution of TiO₂-Tf showing excellent uptake intumors 24 h post injection.

FIG. 38A, FIG. 38B and FIG. 38C depict a schematic and images oftitanocene loaded lipid-micellar nanoparticles. (FIG. 38A) Schematic oflipid micellar nanoparticle with titanocene dichloride and VLA-4 homingligands. (FIG. 38B) TEM image of micelles alone. Scale bar, 50 nm. (FIG.38C) TEM image of micelle incorporated with Tc in the membrane as wellas center. Scale bar, 50 nm.

FIG. 39 depicts MTS cell viability assay (48 h) demonstrating higherdegree of cell death when treated with micelle+Tc+FDG, in both 5TGM andU266 MM cell lines. 1. Untreated. 2. Positive control (Staurosporine).3. FDG 31 MBq/0.1 ml. 4. Micelle. 5. Micelle+Tc. 6. Micelle+FDG. 7.Micelle+Tc+FDG.

FIG. 40A, FIG. 40B, FIG. 40C, FIG. 40D and FIG. 40E depict MM animalmodels. (FIG. 40A) Ventral and (FIG. 40B) dorsal view of bioluminescenceimaging of NSG mice injected with MM1.S luciferase expressing cellsshowing the proliferation of MM in the joints and skeletal tissue. (FIG.40C) In vivo fluorescence imaging of GFP-5TGM1 Cells in KaLwRij miceshowing in the spleen and bone marrow of the femur. (FIG. 40D) MetabolicPET imaging of 5TGM1 distribution in KaLwRij mice using ¹⁸FDG. (FIG.40E) PET imaging of VLA-4 receptor positive 5TGM1 cells in KaLwRij miceusing ⁶⁴Cu-CB-TE1A1P-LLP2A. K, kidney; H, heart; S, spleen; T, MM tumor.

FIG. 41A and FIG. 41B depict graphs showing the pharmacokinetics andbiodistribution of Tc loaded VLA-4 targeted nanomicelles. (FIG. 41A)Pharmacokinetics of micelle+Tc using ICP OES. Half-life is 123.4 min.(FIG. 41B) Biodistribution of targeted micelle+Tc in vivo showinghighest uptake and retention in tumors (5TGM subcutaneous xenografts) 24h post injection.

FIG. 42A, FIG. 42B and FIG. 42C depict in vivo CRIT using 5tGM xenograftMM mouse model. (FIG. 42A) Kaplan-Meier survival curves showing increasein median survival by 7 d in targeted CRIT. (FIG. 42B) Ex vivofluorescence image of untreated tumor using filter for GFP (Ex/Em:488/535 nm). (FIG. 42C) Ex vivo fluorescence image of treated tumor.

DETAILED DESCRIPTION OF THE INVENTION

Photodynamic therapy (PDT) is based on the use of light-sensitivemolecules. When light-sensitive molecules are activated by light atspecific wavelengths, they cause a variety of active forms of oxygen tobe created, the main one of which is singlet oxygen. The processinvolves absorption of photons by the light-sensitive molecule toproduce an excited state which, ultimately, transfers its energy toavailable surrounding oxygen to produce a molecular excited state ofoxygen in the singlet stage. This reaction is common to essentially alllight-sensitive molecules currently being studied for possibleapplications in PDT. The formation of singlet oxygen in cell membranes,cytoplasm or organelles results in peroxidative reactions that causecell damage and death. Administration of the light-sensitive molecule,followed, at the appropriate time, by light treatment using a wavelengththat activates the light-sensitive molecule, may result in effectiveablation of the targeted tissue. However, PDT is limited to superficialtissue and is unable to penetrate depper into tissues.

A method of administering Cerenkov-radiation induced therapy (CRIT) thatovercomes the limitation of use in deeper tissues has been developed.Using a method of the invention, it is possible to perform CRIT ondeeper tissues by using Cerenkov radiation (CR)-emitting radionuclidesto activate at least one radiation-sensitive molecule therebyeliminating the need for an external light source. Advantageously,CR-induced therapy of the invention may allow the amount of radiationadministered to be 100-fold less than the currently administered amountof radiation in clinical and nuclear radiotherapy. By activatingradiation-sensitive molecules using CR-emitting radionuclides, methodsof the invention also provide means for imaging a tumor and monitoringtumor progression in a subject.

I. Components of CR-PDT

A. Composition

In an aspect, the invention encompasses a composition comprising atleast one electromagnetic radiation-sensitive molecule. As used herein,“electromagnetic radiation” and “radiation” are used interchangeably. Inan embodiment, a composition may comprise at least two electromagneticradiation-sensitive molecules. For example, a composition may comprise2, 3, 4, or 5 or more electromagnetic radiation-sensitive molecules.Electromagnetic radiation may include radiowaves, microwaves,near-infrared radiation, infrared radiation, visible light, ultravioletradiation, X-ray and gamma rays. In a specific embodiment, theelectromagnetic radiation is light. Non-limiting examples of light mayinclude ultraviolet (UV), visible, infrared, and near infrared (NIR).

In an embodiment, an electromagnetic radiation-sensitive molecule may bea light-sensitive molecule. A light-sensitive molecule may be aphotosensitizer, a photocatalyst, and/or a photoinitiator. Alight-sensitive molecule may be both a photosensitizer and aphotocatalyst. Additionally, a light-sensitive molecule may be both aphotosensitizer and a phototinitiator. As used herein, the term“photosensitizer” refers to a molecule capable of the photochemicalconversion of an irradiating energy into radical and cytotoxic species.A photosensitizer may also be a photoinitiator or a photocatalyst. Asused herein a “photoinitiator” is a chemical compound that decomposesinto free radicals when exposed to electromagnetic radiation. Allphotoinitiators have bonds that cleave via photolysis. A photoinitiatorconverts absorbed electromagnetic radiation into chemical energy in theform of initiating species, e.g. free radicals or cations. In a specificembodiment, a photoinitiator converts light inot chemical energy.Non-limiting examples of light may include UV, visible, near infraredand infrared. As used herein a “photocatalyst” is a substance which canmodify the rate of chemical reaction using electromagnetic radiation,preferably light. Generally speaking, photocatalysis is a reaction whichuses light to activate a substance which modifies the rate of a chemicalreaction without being involved itself.

In an embodiment, a composition may comprise one or morephotosensitizers. In another embodiment, a composition may comprise oneor more photoinitiators. In still another embodiment, a composition maycomprise one or more photocatalysts. In a different embodiment, acomposition may comprise one or more photosensitizer and one or morephotoinitiators. In another different embodiment, a composition maycomprise one or more photosensitizers and one or more photocatalysts. Instill another different embodiment, a composition may comprise one ormore photosensitizers, one or more photoinitiators, and one or morephotocatalysts. In still yet another different embodiment, a compositionmay comprise one or more photocatalysts and one or more photoinitiators.In a specific embodiment, a composition may comprise a photosensitizerand a photoinitiator. In another specific embodiment, a composition maycomprise a photosensitizer and a photocatalyst. In still anotherspecific embodiment, a composition may comprise a photosensitizer, aphotoinitiator, and a photocatalyst. In still yet another specificembodiment, a composition may comprise a photocatalyst and aphotoinitiator.

A variety of molecules may be used as photosensitizers. Non-limitingexamples of photosensitizers include pyrrole derived macrocycliccompounds, porphyrins, chlorins, bacteriochlorins, isobacteriochlorins,phthalocyanines, naphthalocyanines, porphycenes, porphycyanines,pentaphyrins, sapphyrins, benzochlorins, chlorophylls, azaporphyrins,the metabolic porphyrinic precusor 5-amino levulinic acid, PHOTOFRIN®,synthetic diporphyrins and dichlorins, phenyl-substituted tetraphenylporphyrins (e.g., FOSCAN® picket fence porphyrins), indium chloridemethyl pyropheophorbide (MV64013™), 3,1-meso tetrakis (o-propionamidophenyl) porphyrin, verdins, purpurins (e.g., tin and zinc derivatives ofoctaethylpurpurin (NT2), and etiopurpurin (ET2)), zincnaphthalocyanines, anthracenediones, anthrapyrazoles,aminoanthraquinone, phenoxazine dyes, chlorins (e.g., chlorin e6, andmono-1-aspartyl derivative of chlorin e6), benzoporphyrin derivatives(BPD) (e.g., benzoporphyrin monoacid derivatives, tetracyanoethyleneadducts of benzoporphyrin, dimethyl acetylenedicarboxylate adducts ofbenzoporphyrin, Diels-Adler adducts, and monoacid ring “a” derivative ofbenzoporphyrin), low density lipoprotein mediated localizationparameters similar to those observed with hematoporphyrin derivative(HPD), sulfonated aluminum phthalocyanine (Pc) (sulfonated AlPc,disulfonated (AlPcS₂), tetrasulfonated derivative, sulfonated aluminumnaphthalocyanines, chloroaluminum sulfonated phthalocyanine (CASP)),phenothiazine derivatives, chalcogenapyrylium dyes cationic selena andtellurapyrylium derivatives, ring-substituted cationic phthalocyanines,pheophorbide alpha, hydroporphyrins (e.g., chlorins and bacteriochlorinsof the tetra(hydroxyphenyl) porphyrin series), phthalocyanines,hematoporphyrin (HP), protoporphyrin, uroporphyrin III, coproporphyrinIII, protoporphyrin IX, 5-amino levulinic acid, pyrromethane borondifluorides, indocyanine green, zinc phthalocyanine, dihematoporphyrin,benzoporphyrin derivatives, carotenoporphyrins, hematoporphyrin andporphyrin derivatives, rose bengal, bacteriochlorin A, epigallocatechin,epicatechin derivatives, hypocrellin B, urocanic acid, indoleacrylicacid, rhodium complexes, etiobenzochlorins, octaethylbenzochlorins,sulfonated Pc-naphthalocyanine, silicon naphthalocyanines,chloroaluminum sulfonated phthalocyanine, phthalocyanine derivatives,iminium salt benzochlorins, and other iminium salt complexes, Merocyanin540, Hoechst 33258, and other DNA-binding fluorochromes, psoralens,acridine compounds, suprofen, tiaprofenic acid, non-steroidalanti-inflammatory drugs, methylpheophorbide-a-(hexyl-ether), and otherpheophorbides, furocoumarin hydroperoxides, Victoria blue BO, methyleneblue, toluidine blue, porphycene compounds described in U.S. Pat. No.5,179,120, indocyanines, semiconductor nanoparticle photosensitizers,and any other photosensitizers noted herein, and any combination of anyor all of the above.

In an embodiment, a photosensitizer may be a fullerene. A fullerene is amolecule composed entirely of carbon, in the form of a hollow sphere,ellipsoid, tube, and many other shapes. Spherical fullerenes are alsocalled buckyballs. Cylindrical fullerenes are called carbon nanotubes orbuckytubes. Types of fullerene may include buckyball clusters,nanotubes, carbon nanobuds, megatubes, polymers, nano“onions”, linked“ball-and-chain” dimers, fullerene rings, and inorganic fullerenes suchas MoS₂, WS₂, TiS₂ and NbS₂.

In a specific embodiment, a photosensitizer may be an inorganicnanoparticle. An inorganic nanoparticle may also be a photocatalyst. Aninorganic nanoparticle may be selected from the group consisting of ZnOnanoparticles, Si nanoparticles, TiO₂ nanoparticles, CdSe nanoparticles,CdS nanoparticles, InP nanoparticles, PbS nanoparticles, PbSenanoparticles, and combinations thereof. In an exemplary embodiment, thephotosensitizer is TiO₂ nanoparticles. Traditional photosensitizersdepend on molecular oxygen to generate cytotoxic singlet oxygen for PDT.However, in solid tumors hypoxic conditions prevail, limiting thetherapeutic efficacy of the photosensitizer. Biocompatible inorganicnanoparticles which generate highly cytotoxic hydroxyl radicals throughoxygen-independent electron-hole pair production are attractivealternatives to conventional photosensitizers. Further, biocompatibleinorganic nanoparticles, are attractive photosensitizers because oftheir large surface area, excellent payload capacity, and highreactivity. Semiconductor nanoparticles such as TiO₂ and ZnO areeffective photocatalysts that are capable of generating singlet oxygenfor killing cancer cells and bacteria (Wang et al, Journal of MaterialsChemistry, 2004, 14: 487). For example, B-chelated TiO₂ nanocompositehas a high efficiency of singlet oxygen generation when irradiated withvisible light (Xu et al, Journal of Photochemistry and Photobiology B:Biology, 2002). TiO₂ is a biocompatible material, which encourages theapplication of TiO₂ nanoparticles as a PDT agent for cancer treatment.Similar to other photosensitizers, semiconductor nanoparticles such asTiO₂ and ZnO only have strong absorption in UV or visible ranges, whichlimits their application in conventional PDT. The presently disclosedmethodology provides a means of circumventing the problem of lightactivation.

A variety of molecules may be used as photoinitiators. Photoinitiatorsmay be divided into classes such as acetophenone, benzyl and benzoincompounds, benzophenone, cationic photoinitiators, and thioxanthones.Non-limiting examples of biocompatible photoinitiators includetitanocene or titanocene dichloride, Irgacure-2959(2-hydroxy-1-[4-(hydroxyethoxy)phenyl]-2-methyl-1-propanone; <313 nm AbsMax), Darocur-2959 (2-hydroxy-1-[4-(hydroxyethoxy)phenyl]-1-propanone;<313 nm Abs Max), Irgacure-184 (1-hydroxycyclohexane acetophenone; 326nm Abs Max), Irgacure-651 (2,2-dymethoxy-2-phenyl acetophenone; 335 nmAbs Max), THX (thioxanthone; 378 nm Abs Max), Eosin Y (514 nm Abs Max),camphorquinone and its derivatives (200-300 nm and 467 nm), BAPO(bisacylphosphine oxide bis(2,4,6-trimethylbenzoyl)phenylphosphineoxide; visible), and HAP (hydroxyalkylphenone; <400 nmAbs Max). In an exemplary embodiment, the photoiniator is titanocene.

In a certain embodiment, a composition of the invention may comprise acoating to eliminate undesirable effects and improve biocompatibility.In its native state, a photosensitizer such as TiO₂ may exhibitconcentration dependent cytotoxity. To eliminate this undesirable effectand improve biocompatibility, a radiation-sensitive molecule may becoated. Non-limiting examples of potential coatings may includepolyethylene glycol (PEG), dextran, pullulan, glycolipid, hyaluronicacid, orosomucoid, heparin, chitosan, pectin, or other polysaccharides.Further, there are numerous methodologies to coat a radiation-sensitivemolecule. Non-limiting examples of methods to coat a radiation-sensitivemolecule may include adsorption, incorporation, copolymerization, orcovalent grafting. In a specific embodiment, a radiation-sensitivemolecule is coated with PEG. In another specific embodiment, aradiation-sensitive molecule is coated with dextran. In an exemplaryembodiment, TiO₂ nanoparticles are coated with PEG. In another exemplaryembodiment, TiO₂ nanoparticles are coated with dextran.

In a specific embodiment, a composition further comprises a targetingagent. A targeting agent may promote targeting of theradiation-sensitive molecule. For example, a radiation-sensitivemolecule may be coated with a targeting agent. Additionally, thetargeting agent may bind a radiation-sensitive molecule with highaffinity. In an embodiment, a photosensitizer is coated with a targetingagent. In another embodiment, a targeting agent binds a photoinitiator.In still another embodiment, a photosensitizer is coated with atargeting agent and the targeting agent binds a photoinitiator with highaffinity.

A targeting agent can have an affinity for a cell, a tissue, a protein,DNA, RNA, an antibody, an antigen, a compound, and the like, that may beassociated with a condition, disease, or related biological event, ofinterest. In a specific embodiment, the targeting agent has affinity fora tumor. In particular, the targeting agent can function to targetspecific DNA, RNA, and/or proteins of interest. In an embodiment, thetargeting agent can include, but is not limited to, polypeptides (e.g.,proteins such as, but not limited to, cell surface receptors andantibodies (monoclonal or polyclonal)), antigens, nucleic acids (bothmonomeric and oligomeric), polysaccharides, sugars, fatty acids,steroids, purines, pyrimidines, ligands, aptamers, small molecules,albumin, or combinations thereof, that have an affinity for a condition,disease, or related biological event or other chemical, biochemical,and/or biological events of the condition, disease, or biological event.In an embodiment, the targeting agent can include: aptamers,sequence-specific DNA oligonucleotides, locked nucleic acids (LNA), andpeptide nucleic acids (PNA), antibodies, and small molecule proteinreceptors. For example, when liver targeting is desired, a compositionmay comprise galactose-containing copolymers which are recognized byhepatocytes. Or, for example, when tumor targeting is desired, acomposition may comprise transferrin which binds to transferrinreceptors which are highly overexpressed on tumors. One of skill in theart will appreciate that various targeting agents may enable targetingof a radiation-sensitive molecule to specific tissue. For example, aradiation-sensitive molecule may be conjugated to antibodies in order toprovide specific delivery of the radiation-sensitive molecule to thesite of a tumor. In an embodiment, a targeting agent may be transferrin.As such, a radiation-sensitive molecule coated with transferrin may betargeted to tumor cells. In an exemplary embodiment, TiO₂ is coated withtransferrin. In another exemplary embodiment, transferrin bindstitanocene. In still another exemplary embodiment, TiO₂ is coated withtransferrin and transferrin binds titanocene with high affinity.

(i) Pharmaceutical Composition

The compositions of the present invention may further comprise a drugcarrier to facilitate drug preparation and administration. Any suitabledrug delivery vehicle or carrier may be used, including but not limitedto a microcapsule, for example a microsphere or a nanosphere (Manome etal., 1994; Saltzman & Fung, 1997), a peptide (U.S. Pat. Nos. 6,127,339and 5,574,172), a glycosaminoglycan (U.S. Pat. No. 6,106,866), a fattyacid (U.S. Pat. No. 5,994,392), a fatty emulsion (U.S. Pat. No.5,651,991), a lipid or lipid derivative (U.S. Pat. No. 5,786,387),collagen (U.S. Pat. No. 5,922,356), a polysaccharide or derivativethereof (U.S. Pat. No. 5,688,931), a nanosuspension (U.S. Pat. No.5,858,410), a polymeric micelle or conjugate (Goldman et al., 1997 andU.S. Pat. Nos. 4,551,482, 5,714,166, 5,510,103, 5,490,840, and5,855,900), and a polysome (U.S. Pat. No. 5,922,545).

Additionally, the composition may be formulated into pharmaceuticalcompositions and administered by a number of different means that maydeliver a therapeutically effective dose. Such compositions may beadministered orally, parenterally, by inhalation spray, rectally,intradermally, transdermally, or topically in dosage unit formulationscontaining conventional nontoxic pharmaceutically acceptable carriers,adjuvants, and vehicles as desired. Topical administration may alsoinvolve the use of transdermal administration such as transdermalpatches or iontophoresis devices. The term parenteral as used hereinincludes subcutaneous, intravenous, intramuscular, or intrasternalinjection, or infusion techniques. Formulation of drugs is discussed in,for example, Hoover, John E., Remington's Pharmaceutical Sciences, MackPublishing Co., Easton, Pa. (1975), and Liberman, H. A. and Lachman, L.,Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y. (1980).

Injectable preparations and formulations for parenteral administrationmay be prepared as described above. Solid dosage forms for oraladministration may include capsules, tablets, pills, powders, andgranules. In such solid dosage forms, the composition is ordinarilycombined with one or more adjuvants appropriate to the indicated routeof administration. If administered per os, the composition can beadmixed with lactose, sucrose, starch powder, cellulose esters ofalkanoic acids, cellulose alkyl esters, talc, stearic acid, magnesiumstearate, magnesium oxide, sodium and calcium salts of phosphoric andsulfuric acids, gelatin, acacia gum, sodium alginate,polyvinylpyrrolidone, and/or polyvinyl alcohol, and then tableted orencapsulated for convenient administration. Such capsules or tablets maycontain a controlled-release formulation as can be provided in adispersion of active composition of the invention in hydroxypropylmethylcellulose. In the case of capsules, tablets, and pills, the dosage formsmay also comprise buffering agents such as sodium citrate, or magnesiumor calcium carbonate or bicarbonate. Tablets and pills may additionallybe prepared with enteric coatings.

Liquid dosage forms for oral administration may include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirscontaining inert diluents commonly used in the art, such as water. Suchcompositions may also comprise adjuvants, such as wetting agents,emulsifying and suspending agents, and sweetening, flavoring, andperfuming agents.

The amount of the composition of the invention that may be combined withthe carrier materials to produce a single dosage of the composition canand will vary depending upon the subject, the radiation-sensitivemolecue, the formulation, and the particular mode of administration.Those skilled in the art will appreciate that dosages may also bedetermined with guidance from Goodman & Goldman's The PharmacologicalBasis of Therapeutics, Ninth Edition (1996), Appendix II, pp. 1707-1711and from Goodman & Goldman's The Pharmacological Basis of Therapeutics,Tenth Edition (2001), Appendix II, pp. 475-493.

In certain embodiments, a composition comprising a radiation-sensitivemolecule of the invention is encapsulated in a suitable vehicle toeither aid in the delivery of the compound to target cells, to increasethe stability of the composition, or to minimize potential toxicity ofthe composition. As will be appreciated by a skilled artisan, a varietyof vehicles are suitable for delivering a composition of the presentinvention. Non-limiting examples of suitable structured fluid deliverysystems may include nanoparticles, liposomes, microemulsions, micelles,dendrimers and other phospholipid-containing systems. Methods ofincorporating compositions into delivery vehicles are known in the art.

In one alternative embodiment, a liposome delivery vehicle may beutilized. Liposomes, depending upon the embodiment, are suitable fordelivery of the a radiation-sensitive molecule of the invention in viewof their structural and chemical properties. Generally speaking,liposomes are spherical vesicles with a phospholipid bilayer membrane.The lipid bilayer of a liposome may fuse with other bilayers (e.g., thecell membrane), thus delivering the contents of the liposome to cells.In this manner, the a radiation-sensitive molecule of the invention maybe selectively delivered to a cell by encapsulation in a liposome thatfuses with the targeted cell's membrane.

Liposomes may be comprised of a variety of different types ofphosolipids having varying hydrocarbon chain lengths. Phospholipidsgenerally comprise two fatty acids linked through glycerol phosphate toone of a variety of polar groups. Suitable phospholids includephosphatidic acid (PA), phosphatidylserine (PS), phosphatidylinositol(PI), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG),phosphatidylcholine (PC), and phosphatidylethanolamine (PE). The fattyacid chains comprising the phospholipids may range from about 6 to about26 carbon atoms in length, and the lipid chains may be saturated orunsaturated. Suitable fatty acid chains include (common name presentedin parentheses) n-dodecanoate (laurate), n-tretradecanoate (myristate),n-hexadecanoate (palmitate), n-octadecanoate (stearate), n-eicosanoate(arachidate), n-docosanoate (behenate), n-tetracosanoate (lignocerate),cis-9-hexadecenoate (palmitoleate), cis-9-octadecanoate (oleate),cis,cis-9,12-octadecandienoate (linoleate), all cis-9, 12,15-octadecatrienoate (linolenate), and allcis-5,8,11,14-eicosatetraenoate (arachidonate). The two fatty acidchains of a phospholipid may be identical or different. Acceptablephospholipids include dioleoyl PS, dioleoyl PC, distearoyl PS,distearoyl PC, dimyristoyl PS, dimyristoyl PC, dipalmitoyl PG, stearoyl,oleoyl PS, palmitoyl, linolenyl PS, and the like.

The phospholipids may come from any natural source, and, as such, maycomprise a mixture of phospholipids. For example, egg yolk is rich inPC, PG, and PE, soy beans contains PC, PE, PI, and PA, and animal brainor spinal cord is enriched in PS. Phospholipids may come from syntheticsources too. Mixtures of phospholipids having a varied ratio ofindividual phospholipids may be used. Mixtures of differentphospholipids may result in liposome compositions having advantageousactivity or stability of activity properties. The above mentionedphospholipids may be mixed, in optimal ratios with cationic lipids, suchas N-(1-(2,3-dioleolyoxy)propyl)-N,N,N-trimethyl ammonium chloride,1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchloarate,3,3′-deheptyloxacarbocyanine iodide,1,1′-dedodecyl-3,3,3′,3′-tetramethylindocarbocyanine perchloarate,1,1′-dioleyl-3,3,3′,3′-tetramethylindo carbocyanine methanesulfonate,N-4-(delinoleylaminostyryl)-N-methylpyridinium iodide, or1,1,-dilinoleyl-3,3,3′,3′-tetramethylindocarbocyanine perchloarate.

Liposomes may optionally comprise sphingolipids, in which spingosine isthe structural counterpart of glycerol and one of the one fatty acids ofa phosphoglyceride, or cholesterol, a major component of animal cellmembranes. Liposomes may optionally, contain pegylated lipids, which arelipids covalently linked to polymers of polyethylene glycol (PEG). PEGsmay range in size from about 500 to about 10,000 daltons.

Liposomes may further comprise a suitable solvent. The solvent may be anorganic solvent or an inorganic solvent. Suitable solvents include, butare not limited to, dimethylsulfoxide (DMSO), methylpyrrolidone,N-methylpyrrolidone, acetronitrile, alcohols, dimethylformamide,tetrahydrofuran, or combinations thereof.

Liposomes carrying the a radiation-sensitive molecule of the invention(i.e., having at least one methionine compound) may be prepared by anyknown method of preparing liposomes for drug delivery, such as, forexample, detailed in U.S. Pat. Nos. 4,241,046, 4,394,448, 4,529,561,4,755,388, 4,828,837, 4,925,661, 4,954,345, 4,957,735, 5,043,164,5,064,655, 5,077,211 and 5,264,618, the disclosures of which are herebyincorporated by reference in their entirety. For example, liposomes maybe prepared by sonicating lipids in an aqueous solution, solventinjection, lipid hydration, reverse evaporation, or freeze drying byrepeated freezing and thawing. In a preferred embodiment the liposomesare formed by sonication. The liposomes may be multilamellar, which havemany layers like an onion, or unilamellar. The liposomes may be large orsmall. Continued high-shear sonication tends to form smaller unilamellarlipsomes.

As would be apparent to one of ordinary skill, all of the parametersthat govern liposome formation may be varied. These parameters include,but are not limited to, temperature, pH, concentration of methioninecompound, concentration and composition of lipid, concentration ofmultivalent cations, rate of mixing, presence of and concentration ofsolvent.

In another embodiment, a composition of the invention may be deliveredto a cell as a microemulsion. Microemulsions are generally clear,thermodynamically stable solutions comprising an aqueous solution, asurfactant, and “oil.” The “oil” in this case, is the supercriticalfluid phase. The surfactant rests at the oil-water interface. Any of avariety of surfactants are suitable for use in microemulsionformulations including those described herein or otherwise known in theart. The aqueous microdomains suitable for use in the inventiongenerally will have characteristic structural dimensions from about 5 nmto about 100 nm. Aggregates of this size are poor scatterers of visiblelight and hence, these solutions are optically clear. As will beappreciated by a skilled artisan, microemulsions can and will have amultitude of different microscopic structures including sphere, rod, ordisc shaped aggregates. In one embodiment, the structure may bemicelles, which are the simplest microemulsion structures that aregenerally spherical or cylindrical objects. Micelles are like drops ofoil in water, and reverse micelles are like drops of water in oil. In analternative embodiment, the microemulsion structure is the lamellae. Itcomprises consecutive layers of water and oil separated by layers ofsurfactant. The “oil” of microemulsions optimally comprisesphospholipids. Any of the phospholipids detailed above for liposomes aresuitable for embodiments directed to microemulsions. The composition ofthe invention may be encapsulated in a microemulsion by any methodgenerally known in the art.

In yet another embodiment, a composition of the invention may bedelivered in a dendritic macromolecule, or a dendrimer. Generallyspeaking, a dendrimer is a branched tree-like molecule, in which eachbranch is an interlinked chain of molecules that divides into two newbranches (molecules) after a certain length. This branching continuesuntil the branches (molecules) become so densely packed that the canopyforms a globe. Generally, the properties of dendrimers are determined bythe functional groups at their surface. For example, hydrophilic endgroups, such as carboxyl groups, would typically make a water-solubledendrimer. Alternatively, phospholipids may be incorporated in thesurface of a dendrimer to facilitate absorption across the skin. Any ofthe phospholipids detailed for use in liposome embodiments are suitablefor use in dendrimer embodiments. Any method generally known in the artmay be utilized to make dendrimers and to encapsulate compositions ofthe invention therein. For example, dendrimers may be produced by aniterative sequence of reaction steps, in which each additional iterationleads to a higher order dendrimer. Consequently, they have a regular,highly branched 3D structure, with nearly uniform size and shape.Furthermore, the final size of a dendrimer is typically controlled bythe number of iterative steps used during synthesis. A variety ofdendrimer sizes are suitable for use in the invention. Generally, thesize of dendrimers may range from about 1 nm to about 100 nm.

B. CR-Emitting Radionuclides

According to the invention, a composition of the invention may beactivated by electromagnetic radiation to generate free radicals. In aspecific embodiment, a composition of the invention may be activated bylow intensity light to generate free radicals. In another specificembodiment, the free radicals may be generated in an oxygen independentfashion. The lack of reliance on molecular oxygen allows activation ofthe composition in hypoxic regions. Many solid tumors have significanthypoxic regions. As such, the present invention overcomes the limitationof the reliance of PDT on molecular oxygen. Low intensity light mayinclude light in the visible spectrum or light in the ultraviolet (UV)spectrum. Generally, light in the visible spectrum comprises wavelengthsfrom about 390 nm to about 700 nm and light in the ultraviolet spectrumcomprises wavelengths from about 100 nm to about 400 nm. In anembodiment, a composition may be activated by light in the UV spectrum.For example, a composition may be activated by light at wavelengths fromabout 250 nm to about 350 nm. Alternatively, a composition may beactivated by light at wavelengths from about 350 nm to about 600 nm, orfrom about 400 nm to about 550 nm. In another embodiment, a compositionmay be activated by Cerenkov radiation (CR)-emitting radionuclides.

Cerenkov radiation (CR) is created by high-energy charged particles thatmomentarily exceed the speed of light in the medium in which theypropagate. As the charged particle travels through the medium, itdisrupts the electromagnetic field of the medium and temporarilydisplaces the electrons in the atoms of the medium. Photons are emittedwhen the displaced electrons return to the ground state after thedisruption has ceased. According to the mechanism of CR, as long asthese positrons have a superluminal speed in a dielectric medium, CRwill be produced until interactions with the medium cause theseparticles to lose kinetic energy to the point that their speed dropsbelow the speed of light in that medium.

A variety of charged particles with the appropriate energy levels canproduce CR. These include high energy x-rays such as those used inradiotherapy and radionuclides that undergo radioactive decay such asβ-particles, Auger electrons, positrons (β+), and α-particles. Ofparticular interest is the use of Positron Emission Tomography (PET)isotopes as the photon source to power in vivo light based imaging andtherapeutic inventions. The Cerenkov light spectrum is continuous, incontrast to fluorescence or emission spectra that have characteristicspectral peaks. The relative intensity is proportional to frequencythus: higher frequencies (ultra-violet/blue) are most intense. Atultraviolet/blue wavelengths, Cerenkov radiation is highly absorbed bytissue components (water, hemogloblin, cytochromes, etc.).

As described herein a wide range of radionuclides may be used in themethods of the present invention. In an embodiment, the radionuclide mayinclude radionuclides except those that are pure gamma rays-emittingradionuclides. In a particular embodiment, the radionuclides may includethose that emit radionuclides that are a, β⁺, β⁻-emitters. Radionuclides(α, β⁺, β⁻, electron capture, etc.) that emit charged particles may besuitable for optical imaging. A radionuclide that produces CR may be aradionuclide following β⁺, β⁻ or electron capture decay. In this regard,a radionuclide employed in the present invention may be a radionuclidethat decays via β⁺ decay such as ¹⁰C, ¹¹C, ¹³O, ¹⁴O. ¹⁵O, ¹²N, ¹³N, ¹⁵F,¹⁸F, ³²Cl, ³³Cl, ³⁴Cl, ⁴³Sc, ⁴⁴Sc, ⁴⁵Ti, ⁵¹Mn, ⁵²Mn ⁵²Fe, ⁵³Fe, ⁵⁵Co,⁵⁶Co, ⁵⁸Co, ⁶¹Cu, ⁶²Cu, ⁶²Zn, ⁶³Zn, ⁶⁴Cu, ⁶⁵Zn, ⁶⁶Ga, ⁶⁶Ge, ⁶⁷Ge, ⁶⁸Ga,⁶⁹Ge, ⁶⁹As, ⁷⁰As, ⁷⁰Se, ⁷¹As, ⁷³Se, ⁷⁴Kr, ⁷⁴Br, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br, ⁷⁷Kr,⁷⁸Br, ⁷⁸Rb, ⁷⁹Rb, ⁷⁹Kr, ⁸¹Rb, ⁸²Rb, ⁸⁴Rb, ⁸⁴Zr, ⁸⁵Y, ⁸⁶Y, ⁸⁷Y, ⁸⁷Zr,⁸⁸Y, ⁸⁹Zr, ⁹²Tc, ⁹³Tc, ⁹⁴Tc, ⁹⁵Tc, ⁹⁵Ru, ⁹⁶Rh, ⁹⁶Rh ⁹⁷Rh, ⁹⁸Rh, ⁹⁹Rh,¹⁰⁰Rh, ¹⁰¹Ag, ¹⁰²Ag, ¹⁰²Rh, ¹⁰³Ag, ¹⁰⁴Ag, ¹⁰⁵Ag, ¹⁰⁶Ag, ¹⁰⁸In, ¹⁰⁹In,¹¹⁰In, ¹¹⁵Sb, ¹¹⁶Sb, ¹¹⁷Sb, ¹¹⁵Te, ¹¹⁶Te, ¹¹⁷Te, ¹¹⁷I, ¹¹⁸I, ¹¹⁸Xe,¹¹⁹Xe, ¹¹⁹I, ¹¹⁹Te, ¹²⁰I, ¹²⁰Xe, ¹²¹Xe, ¹²¹I, ¹²²I, ¹²³Xe, ¹²⁴I, ¹²⁶I,¹²⁸I, ¹²⁹La, ¹³⁰La, ¹³¹La, ¹³²La, ¹³³La, ¹³⁵La, ¹³⁶La, ¹⁴⁰Sm, ¹⁴¹Sm,¹⁴²Sm, ¹⁴⁴Gd, ¹⁴⁵Gd, ¹⁴⁵Eu, ¹⁴⁶Gd, ¹⁴⁶Eu, ¹⁴⁷Eu, ¹⁴⁷Gd, ¹⁴⁸Eu, ¹⁵⁰Eu,¹⁹⁰Au, ¹⁹¹Au, ¹⁹²Au, ¹⁹³Au, ¹⁹⁸Au, ¹⁹⁹Au, ¹⁹³Tl, ¹⁹⁴Tl, ¹⁹⁴Au, ¹⁹⁵Tl,¹⁹⁶Tl, ¹⁹⁷Tl, ¹⁹⁸Tl, ²⁰⁰Tl, ²⁰⁰Bi, ²⁰²Bi, ²⁰³Bi, ²⁰⁵Bi or ²⁰⁶Bi, aradionuclide that decays via β⁻ decay such as ³H, ¹⁴C, ³⁵S, ³²P, ¹³I,⁵⁹Fe, ⁶⁰Co, ⁶⁷Cu, ⁸⁹Sr, ⁹⁰Sr, ⁹⁰Y, ⁹⁹Mo, ¹³³Xe, ¹³⁷Cs, ¹⁵³Sm, ¹⁷⁷Lu or¹⁸⁶Re, or a radionuclide that decays via electron capture such as ¹¹¹In,¹²³I, ¹²⁵I, ²⁰¹Tl, ⁶⁷Ga, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁶²Zn or ⁸²Sr. Mostspecifically, it may be ¹⁸F, ¹¹C, ¹³N, ¹⁵O, ⁶⁰Cu, ⁶⁴Cu, ⁶⁷Cu, ¹²⁴I,⁶⁸Ga, ⁵²Fe, ⁵⁸Co, ³H, ¹⁴C, ³⁵S, ³²P, ¹³¹I, ⁵⁹Fe, ⁶⁰Co, ⁸⁹Sr, ⁹⁰Sr, ⁹⁰Y,⁹⁹Mo, ¹³³Xe, ¹³⁷Cs, ¹⁵³Sm, ¹⁷⁷Lu, ¹⁸⁶Re, ¹²³I, ¹²⁵I, ²⁰¹Ti or ⁶⁷Ga, butis not limited thereto. Since other radionuclides that do not decay viaβ⁺, β⁻ or electron capture can also emit light, they may also be used asthe radionuclide of the present disclosure if they produce CR. In aspecific embodiment, the CR-emitting radionuclides are selected from thegroup consisting of ¹⁸F, ¹⁸F-FDG, ⁶⁴Cu, ⁹⁰Y, ¹²⁴I, and ⁸⁹Zr.

In an embodiment, the composition comprising at least oneradiation-sensitive molecule and the radionuclide can be designed tohave an affinity towards the same target or similar target. In anotherembodiment, the composition comprising at least one radiation-sensitivemolecule and a targeting agent may optionally include a radionuclide ina probe. The radiation-sensitive molecule may be associated with (e.g.,bonded, form a complex with, and the like) the radionuclide directly orindirectly (e.g., via a chemical or biochemical linking group ofcompound), many of which are known in the art. In an embodiment, theradiation-sensitive molecule and radionuclide may be positioned so thatthe optical energy emitted from the radionuclide is maximized. In anembodiment, the probe can be configured that upon interaction with thetarget, the probe undergoes a change so that the radiation-sensitivemolecule and the radionuclide are brought into proximity to maximize theenergy emitted by the radiation-sensitive molecule. Activation of theprobe only upon contact with the targeted tissue may limit toxicityassociated with off target activity.

II. Methods of Using CRIT

In an aspect, the invention provides a method for administeringCerenkov-radiation induced therapy (CRIT) to a target tissue in asubject. The method comprises administering to the subject an effectiveamount of a composition comprising at least one radiation-sensitivemolecule and administering to the subject an amount of a Cerenkovradiation (CR)-emitting radionuclide effective to activate theradiation-sensitive molecule, thereby administering CRIT to the targettissue in the subject.

In another aspect, the present invention provides a method of detectinga tumor in a subject. The method comprises administering to the subjectan effective amount of a composition comprising at least oneradiation-sensitive molecule and an amount of a Cerenkov radiation(CR)-emitting radionuclide effective to activate the radiation-sensitivemolecule, and subsequently imaging the subject for a signal, wherein asignal indicates detection of the tumor.

In yet another aspect, the invention provides a method for monitoring aresponse to treatment in a subject. The method comprises administeringto the subject an effective amount of a composition comprising at leastone radiation-sensitive molecule and an amount of a CR-emittingradionuclide effective to activate the radiation-sensitive molecule,imaging the subject for a signal corresponding to theradiation-sensitive molecule, repeating the aforementioned method at alater time, and subsequently comparing the images, wherein a change insignal corresponding to the radiation-sensitive molecule indicates aresponse to treatment.

In still yet another aspect, the invention provides a method fortreating, stabilizing and/or preventing cancer and associated diseasesin a subject. The method comprises administering to the subject aneffective amount of a composition comprising at least oneradiation-sensitive molecule and an amount of a Cerenkov radiation(CR)-emitting radionuclide effective to activate the radiation-sensitivemolecule, thereby treating, stabilizing and/or preventing the cancer orthe associated diseases. By “treating, stabilizing, or preventingcancer” is meant causing a reduction in the size of a tumor or in thenumber of cancer cells, slowing or preventing an increase in the size ofa tumor or cancer cell proliferation, increasing the disease-freesurvival time between the disappearance of a tumor or other cancer andits reappearance, preventing an initial or subsequent occurrence of atumor or other cancer, or reducing an adverse symptom associated with atumor or other cancer. In a desired embodiment, the percent of tumor orcancerous cells surviving the treatment is at least 20, 40, 60, 80, or100% lower than the initial number of tumor or cancerous cells, asmeasured using any standard assay (e.g., caspase assays, TUNEL and DNAfragmentation assays, cell permeability assays, and Annexin V assays).Desirably, the decrease in the number of tumor or cancerous cellsinduced by administration of CRIT of the invention is at least 2, 5, 10,20, or 50-fold greater than the decrease in the number of non-tumor ornon-cancerous cells. Desirably, the methods of the present inventionresult in a decrease of 20, 40, 60, 80, or 100% in the size of a tumoror in the number of cancerous cells, as determined using standardmethods. Desirably, at least 20, 40, 60, 80, 90, or 95% of the treatedsubjects have a complete remission in which all evidence of the tumor orcancer disappears. Desirably, the tumor or cancer does not reappear orreappears after at least 5, 10, 15, or 20 years.

In each of the foregoing embodiments, the composition comprising atleast one radiation-sensitive molecule may further comprise a targetingagent. In each of the foregoing embodiments, the composition comprisingat least one radiation-sensitive molecule may comprise tworadiation-sensitive molecules. In each of the foregoing embodiments, thecomposition comprising at least one radiation-sensitive molecule maycomprise at least two radiation-sensitive molecules. In each of theforegoing embodiments, the composition comprising at least tworadiation-sensitive molecule may comprise a photosensitizer and aphotoinitiator. In each of the foregoing embodiments, the compositioncomprising at least one radiation-sensitive molecule may comprise aphotosensitizer. In each of the foregoing embodiments, the compositioncomprising at least one radiation-sensitive molecule may comprise aphotoinitiator. In each of the foregoing embodiments, thephotosensitizer may be TiO₂ and the photoinitiator may be titanocene.

Suitable subjects include, but are not limited to, a human, a livestockanimal, a companion animal, a lab animal, and a zoological animal. Asubject may or may not be known to have a tumor. In one embodiment, thesubject may be a rodent, e.g. a mouse, a rat, a guinea pig, etc. Inanother embodiment, the subject may be a livestock animal. Non-limitingexamples of suitable livestock animals may include pigs, cows, horses,goats, sheep, llamas and alpacas. In yet another embodiment, the subjectmay be a companion animal. Non-limiting examples of companion animalsmay include pets such as dogs, cats, rabbits, and birds. In yet anotherembodiment, the subject may be a zoological animal. As used herein, a“zoological animal” refers to an animal that may be found in a zoo. Suchanimals may include non-human primates, large cats, wolves, and bears.In a preferred embodiment, the animal is a laboratory animal.Non-limiting examples of a laboratory animal may include rodents,canines, felines, and non-human primates. In another preferredembodiment, the subject is a human.

A. Method of Administering CRIT

In an aspect, the invention provides a method for administering CRIT toa target tissue in a subject. The method comprises administering to thesubject an effective amount of a composition comprising at least oneradiation-sensitive molecule and an amount of a Cerenkov radiation(CR)-emitting radionuclide effective to activate the radiation-sensitivemolecule, thereby administering CRIT to the target tissue in thesubject. In an embodiment where the radiation-sensitive molecule isactivated by X-rays, the invention also provides a method foradministering radiotherapy to a target tissue in a subject.

In CRIT, one or more (e.g., amount and/or type) radionuclides and one ormore radiation-sensitive molecules (e.g., amount and/or type) areintroduced into the subject. Subsequently, low energy photons generatedby the one or more radionuclide are absorbed by the radiation-sensitivemolecules activating the radiation-sensitive molecules thereby causing avariety of active forms of oxygen to be created, the main one of whichis singlet oxygen. It is advantageous for the radiation-sensitivemolecules to absorb energy from the radionuclide so that an outsideenergy source is not needed to excite the radiation-sensitive molecule,since the outside or external energy has limited tissue depthpenetration resulting in limited radiation-sensitive moleculesactivation in deep tissue. Thus, in an embodiment, the method used doesnot need the use of an external, outside, or another source of energy toexcite the radiation-sensitive molecules since the radionuclides excitethe radiation-sensitive molecules. As such, Cerenkov radiation serves asa tissue depth-independent light source for CRIT.

According to the invention, if the radiation-sensitive molecule and theradionuclide are present at the area of the target, the absorption ofphotons generated by the radionuclide by the radiation-sensitivemolecule produces an excited state which, ultimately, transfers itsenergy to available surrounding oxygen to produce a molecular excitedstate of oxygen in the singlet stage. The formation of singlet oxygen incell membranes, cytoplasm or organelles results in peroxidativereactions that cause cell damage and death. As such, the methods of theinvention may be used to treat a disease associated with the targettissue. The terms “treat”, “treating” or “treatment” include prevention,attenuation, reversal, or improvement in at least one symptom or sign ofsymptoms associated with the disease. In one embodiment, the targettissue may be a tumor. As such, the methods of the invention may be usedto treat a tumor derived from a neoplasm or a cancer. The neoplasm maybe malignant or benign, the cancer may be primary or metastatic; theneoplasm or cancer may be early stage or late stage. Non-limitingexamples of neoplasms or cancers that may be treated include acutelymphoblastic leukemia, acute myeloid leukemia, adrenocorticalcarcinoma, AIDS-related cancers, AIDS-related lymphoma, anal cancer,appendix cancer, astrocytomas (childhood cerebellar or cerebral), basalcell carcinoma, bile duct cancer, bladder cancer, bone cancer, brainstemglioma, brain tumors (cerebellar astrocytoma, cerebralastrocytoma/malignant glioma, ependymoma, medulloblastoma,supratentorial primitive neuroectodermal tumors, visual pathway andhypothalamic gliomas, breast cancer, bronchial adenomas/carcinoids,Burkitt lymphoma, carcinoid tumors (childhood, gastrointestinal),carcinoma of unknown primary, central nervous system lymphoma (primary),cerebellar astrocytoma, cerebral astrocytoma/malignant glioma, cervicalcancer, childhood cancers, chronic lymphocytic leukemia, chronicmyelogenous leukemia, chronic myeloproliferative disorders, coloncancer, cutaneous T-cell lymphoma, desmoplastic small round cell tumor,endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma inthe Ewing family of tumors, extracranial germ cell tumor (childhood),extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancers(intraocular melanoma, retinoblastoma), gallbladder cancer, gastric(stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinalstromal tumor, germ cell tumors (childhood extracranial, extragonadal,ovarian), gestational trophoblastic tumor, gliomas (adult, childhoodbrain stem, childhood cerebral astrocytoma, childhood visual pathway andhypothalamic), gastric carcinoid, hairy cell leukemia, head and neckcancer, hepatocellular (liver) cancer, Hodgkin lymphoma, hypopharyngealcancer, hypothalamic and visual pathway glioma (childhood), intraocularmelanoma, islet cell carcinoma, Kaposi sarcoma, kidney cancer (renalcell cancer), laryngeal cancer, leukemias (acute lymphoblastic, acutemyeloid, chronic lymphocytic, chronic myelogenous, hairy cell), lip andoral cavity cancer, liver cancer (primary), lung cancers (non-smallcell, small cell), lymphomas (AIDS-related, Burkitt, cutaneous T-cell,Hodgkin, non-Hodgkin, primary central nervous system), macroglobulinemia(Waldenström), malignant fibrous histiocytoma of bone/osteosarcoma,medulloblastoma (childhood), melanoma, intraocular melanoma, Merkel cellcarcinoma, mesotheliomas (adult malignant, childhood), metastaticsquamous neck cancer with occult primary, mouth cancer, multipleendocrine neoplasia syndrome (childhood), multiple myeloma/plasma cellneoplasm, mycosis fungoides, myelodysplastic syndromes,myelodysplastic/myeloproliferative diseases, myelogenous leukemia(chronic), myeloid leukemias (adult acute, childhood acute), multiplemyeloma, myeloproliferative disorders (chronic), nasal cavity andparanasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma,non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer,oropharyngeal cancer, osteosarcoma/malignant fibrous histiocytoma ofbone, ovarian cancer, ovarian epithelial cancer (surfaceepithelial-stromal tumor), ovarian germ cell tumor, ovarian lowmalignant potential tumor, pancreatic cancer, pancreatic cancer (isletcell), paranasal sinus and nasal cavity cancer, parathyroid cancer,penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma,pineal germinoma, pineoblastoma and supratentorial primitiveneuroectodermal tumors (childhood), pituitary adenoma, plasma cellneoplasia, pleuropulmonary blastoma, primary central nervous systemlymphoma, prostate cancer, rectal cancer, renal cell carcinoma (kidneycancer), renal pelvis and ureter transitional cell cancer,retinoblastoma, rhabdomyosarcoma (childhood), salivary gland cancer,sarcoma (Ewing family of tumors, Kaposi, soft tissue, uterine), Sezarysyndrome, skin cancers (nonmelanoma, melanoma), skin carcinoma (Merkelcell), small cell lung cancer, small intestine cancer, soft tissuesarcoma, squamous cell carcinoma, squamous neck cancer with occultprimary (metastatic), stomach cancer, supratentorial primitiveneuroectodermal tumor (childhood), T-Cell lymphoma (cutaneous),testicular cancer, throat cancer, thymoma (childhood), thymoma andthymic carcinoma, thyroid cancer, thyroid cancer (childhood),transitional cell cancer of the renal pelvis and ureter, trophoblastictumor (gestational), enknown primary site (adult, childhood), ureter andrenal pelvis transitional cell cancer, urethral cancer, uterine cancer(endometrial), uterine sarcoma, vaginal cancer, visual pathway andhypothalamic glioma (childhood), vulvar cancer, Waldenströmmacroglobulinemia, and Wilms tumor (childhood). In an embodiment, theneoplasm or cancer is selected from the group consisting of pancreaticcancer, fibrosarcoma, multiple myeloma and lung cancer. In specificembodiments, the neoplasm or cancer is pancreatic cancer. In otherspecific embodiments, the neoplasm or cancer is fibrosarcoma. In stillother specific embodiments, the neoplasm or cancer is multiple myeloma.In a different embodiment, the neoplasm or cancer is lung cancer.

In another embodiment, the methods of the invention may be used to treata disease associated with diseased and/or inflamed tissues. For example,the new methods may be useful for the treatment of ophthalmologicdisorders such as age-related macular degeneration, diabeticretinopathy, and choroidal neovascularization; dermatological disorderssuch as acne, psoriasis and scleroderma; gynecological disorders such asdysfunctional uterine bleeding; urological disorders such as condylomavirus; cardiovascular disorders such as restenosis, intimal hyperplasia,and atherosclerotic plaques; hemangioma; autoimmune diseases such asarthritis; hyperkeratotic diseases; and for hair removal. Normal ordiseased tissue on any part of the body can be treated or studies withCRIT; thus, normal or abnormal conditions of the hematological system,the lymphatic reticuloendothelial system, the nervous system, theendocrine and exocrine system, the skeletomuscular system includingbone, connective tissue, cartilage and skeletal muscle, the pulmonarysystem, the gastrointestinal system including the liver, thereproductive system, the immune system, the cardiovascular system, theurinary system, the ocular system, and the auditory and olfactorysystems may be treated using the new methods.

In certain aspects, the methods of the invention may further compriseadministering therapeutic agents for neoplasms and cancer. Suitabletherapeutic agents for neoplasms and cancers are known in the art, andwill depend upon the type and stage of cancer. Summaries of cancerdrugs, including information regarding approved indications, may befound via the National Cancer Institute at the National Institutes ofHealth (www.cancer.gov/cancertopics/druginfo/alphalist) and the FDAApproved Drug Product database(www.accessdata.fda.gov/scripts/cder/drugsatfda/).

An exemplary embodiment of the present disclosure includes a method ofreducing the amount of radiation administered to a subject. According tothe invention, a CR-emitting radionuclide may be administered at about a100-fold lower dose than standard treatment. For example, a CR-emittingradionuclide may be administered at about a 2-fold, about a 5-fold,about a 10-fold, about a 20-fold, about a 30-fold, about a 40-fold,about a 50-fold, about a 60-fold, about a 70-fold, about an 80-fold,about a 90-fold or about 100-fold lower dose than standard treatment.Alternatively, a CR-emitting radionuclide may be administered at greaterthan 100-fold lower dose than standard treatment. In a specificembodiment, a CR-emitting radionuclide may be administered at about a2-fold. A lower dose of radionuclide may reduce radiotoxicity to asubject. For example, a standard dose of ¹⁸F-FDG may be about 10 mCi,thus the present methodology may use a dose of about 5 to about 0.01mCi. For example, using the methodology disclosed herein the dose of¹⁸F-FDG may be about 0.15, about 0.2, about 0.25, about 0.3, about 0.35,about 0.4, about 0.45, about 0.5, about 0.55, about 0.6, about 0.65,about 0.7, about 0.75, about 0.8, about 0.85, about 0.9, about 0.95,about 1, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about1.6, about 1.7, about 1.8, about 1.9, about 2, about 2.5, about 3, about3.5, about 4, about 4.5, or about 5 mCi.

B. Method of Detecting and Monitoring a Tumor

In another aspect, the present invention provides a method of detectinga tumor in a subject. The method comprises administering to the subjectan effective amount of composition comprising at least oneradiation-sensitive molecule and an amount of a Cerenkov radiation(CR)-emitting radionuclide effective to activate the radiation-sensitivemolecule, and subsequently imaging the subject for a signalcorresponding to the radiation-sensitive molecule, wherein a signalcorresponding to the radiation-sensitive molecule indicates detection ofthe tumor.

In yet another aspect, the invention provides a method for monitoring atumor in a subject. The method comprises administering to the subject aneffective amount of a composition comprising at least oneradiation-sensitive molecule and an amount of a CR-emitting radionuclideeffective to activate the radiation-sensitive molecule, imaging thesubject for a signal corresponding to the radiation-sensitive molecule,repeating the aforementioned method at a later time, and subsequentlycomparing the images, wherein a change in signal corresponding to theradiation-sensitive molecule indicates a change in tumor.

The invention comprises, in part, imaging a subject. Non-limitingexamples of modalities of imaging may include magnetic resonance imaging(MRI), ultrasound (US), computed tomography (CT), Positron EmissionTomography (PET), Single Photon Emission Computed Tomography (SPECT),and optical imaging (01, bioluminescence and fluorescence). Radioactivemolecular probes are traditionally imaged with PET, SPECT or gamma (γ)cameras, by taking advantage of the capability of these imagingmodalities to detect the high energetic γ rays. In contrast, OIgenerally detects low energy lights (visible or near-infrared lights)emitted from bioluminescence or fluorescence probes. Each modality hasits own advantages and disadvantages. For instance, nuclear imagingmodalities such as PET have high sensitivity and excellentquantification capability but suffer from poor spatial resolution, whichis confined to millimeter range. On the other hand, MRI featuressubmillimeter spatial resolution but is limited by low sensitivity andthe high cost of instrumentation. Much less expensive and more widelyavailable than PET and MRI, traditional 01 also features highsensitivity, short scanning time, and relatively high throughput, yetits potential has been mostly constrained to preclinical studies becauseof low penetration and high scattering of optical signals in livingtissues. The present invention overcomes these limitations allowingoptical imaging to become a new mode of in vivo imaging besides PET andSPECT. In an exemplary embodiment, the imaging is optical imaging.

Compared to conventional fluorescence and bioluminescence opticalimaging, radioactive optical imaging (01) has some unique properties.The continued emission wavelength of radioactive 01 allows monitoringand imaging of a radionuclide at different wavelengths, which is asignificant advantage over the conventional optical imaging modalities.And the radioactive 01 signal generated by a radionuclide does notrequire an excitation light and is always on, which is different fromfluorescence and bioluminescence probes, which typically need an outsidesource of energy and which may produce unwanted and complicating opticalsignals from other sources (e.g., skin). In an embodiment, the signalgenerated by a radionuclide can be used to excite a radiation-sensitivemolecule so the radiation-sensitive molecule can activate without therequirement of an excitation light.

As mentioned above, an exemplary embodiment of the present disclosureincludes a method of imaging a target within a subject using aradionuclide and a composition comprising at least oneradiation-sensitive molecule. Initially, one or more (e.g., amountand/or type) radionuclides and one or more radiation-sensitive molecules(e.g., amount and/or type) may be introduced into the subject.Subsequently, low energy photons generated by the one or moreradionuclide are detected as a signal(s) and/or the radiation-sensitivemolecule absorbs the energy from the radionuclide and then theradiation-sensitive molecule emits a signal. In an exemplary embodiment,the signal is an optical signal. It is advantageous for theradiation-sensitive molecule to absorb energy from the radionuclide sothat an outside energy source is not needed to excite theradiation-sensitive molecule, since the outside or external energysource can also cause other background signals (e.g., optical signals)to be generated, thereby interfering with the signal of interest. Thus,in an embodiment, the method used does not need the use of an external,outside, or another source of energy to excite the radiation-sensitivemolecule since the radionuclides excite the radiation-sensitivemolecule.

According to the invention, if the composition comprising at least oneradiation-sensitive molecule and the radionuclide are present at thearea of the target, the radiation-sensitive molecule should emit energyassociated with a signal that corresponds to the radiation-sensitivemolecule. The various signals can be separated based on wavelengthand/or intensity to determine the area where the signal from theradiation-sensitive molecule is derived. After the signal correspondingto the radiation-sensitive molecule is obtained, the signal or datacorresponding to the detected signal can be processed to provide animage of the target or the area where the target is located. In anembodiment, the image can be a planar image or can be a 3-dimensionalimage of the target. In particular, the signal can be used to identifyan area from which the signal is produced, where the area corresponds tothe location of the target. For example, measuring a signalcorresponding to the radiation-sensitive molecule that is concentratedin a specific area or location is indicative that the target is presentat the location of the origin of the signal.

Once the signal corresponding to the radiation-sensitive molecule isobtained, the status of the condition or disease can be evaluated ormonitored by comparing the image with one or more previous images andone or more subsequent images. In an embodiment, a change in signalindicates a response to treatment. A decrease in signal may indicate adecrease in disease. For example, a decrease in signal may indicate adecrease in tumor size and therefore tumor regression. Alternatively, anincrease in signal may indicate an increase in disease. For example, anincrease in signal may indicate an increase in tumors size and thereforetumor progression.

The term “signal” as used herein, refers to a signal derived from aradioactive substance, a radiation-sensitive molecule, a light-sensitivemolecule, a photosensitizer, a photoinitiator, a photocatalyst etc. thatcan be detected and quantitated with regards to its frequency and/oramplitude. The signal may be an optical signal. The signal can begenerated from one or more radionuclides, radiation-sensitive molecules,or probes of the present disclosure. In an embodiment, the signal mayneed to be the sum of each of the individual signals. In an embodiment,the signal can be generated from a summation, an integration, or othermathematical process, formula, or algorithm, where the signal is fromone or more radionuclides, radiation-sensitive molecules, probes, or thelike. In an embodiment, the summation, the integration, or othermathematical process, formula, or algorithm can be used to generate thesignal so that the signal can be distinguished from background noise andthe like. It should be noted that signals other than the signal ofinterest can be processed and/or obtained in a similar manner as that ofthe signal of interest.

As noted above, embodiments of the present disclosure include a methodfor imaging a target within a subject. In an embodiment, the system caninclude a detection system and a signal processing system. In anembodiment, the detection system can be configured to detect low energyphotons generated by one or more radionuclides as signals within thesubject. In an embodiment, the signal processing system is configured toprovide images based upon the signal. As described above, the signal canbe processed to produce an image. As described herein, the location ofthe target can be obtained using the data or information correspondingto the signal. Additional details are provided in the Examples.

In an embodiment, the system can be an in vivo imaging system that canbe used to visualize molecular events in an organism by detectingemitted photons. In an embodiment, the system can be an opticaldetection system wherein the optical detection system includes anoptical fiber system (e.g., optical fiber, optics for focusing and ordirection the optical energy). The optical signal is directed using theoptical fiber system to a charge-coupled device camera (CCD camera) thatare often utilized for capturing images and converting them into digitalvalues to produce an image.

Embodiments of the methods of the present disclosure may be useful forradioactive optical imaging in cancer imaging and in imaging of otherdiseases. Radioactive optical imaging can be used in the detection,characterization and/or determination of the localization of a diseaseranging from early to late stage disease. Radioactive optical imaginghas, furthermore, utility in staging a disease, i.e., determining theseverity of a disease, monitoring the progression (worsening) of adisease, and/or monitoring the regression (improvement). Radioactiveoptical imaging can also be used in the prognosis of a disease ordisease conditions. Radioactive optical imaging could be very useful forcancer imaging.

In addition, radioactive optical imaging has further utility in imagingdiseases that are characterized by inflammation processes such asrheumatoid arthritis, whereby the presence and location of inflammationcan be imaged; cardiovascular diseases including atherosclerosis,ischemia, stroke, or thromboses, whereby plaques, areas at risk foracute occlusion as well as areas of hypoxia can be imaged; infectiousdiseases, whereby areas inflicted with bacterial, viral, fungal,parasitic pathogens can be imaged. Radioactive optical imaging mightalso be useful for imaging immune cells to aid diagnosing immunologicaldiseases and for neuroimaging to aid diagnosing neurodegenerativediseases.

The term “molecular imaging”, as used herein, relates to the in-vivocharacterization and measurement of biologic processes and pathways atthe cellular and molecular levels.

The term “optical imaging”, as used herein, relates to the generation ofimages by using photons in a wavelength range (e.g., ultraviolet toinfrared).

The term “radioactive optical imaging” and “radioactive molecularoptical imaging” as used herein, relate to the detection of opticalsignals generated by radionuclides and are used interchangeably.

C. Administration

Pharmaceutical compositions for effective administration aredeliberately designed to be appropriate for the selected mode ofadministration, and pharmaceutically acceptable excipients such ascompatible dispersing agents, buffers, surfactants, preservatives,solubilizing agents, isotonicity agents, stabilizing agents and the likeare used as appropriate. Remington's Pharmaceutical Sciences, MackPublishing Co., Easton Pa., 16Ed ISBN: 0-912734-04-3, latest edition,incorporated herein by reference in its entirety, provides a compendiumof formulation techniques as are generally known to practitioners.

In an embodiment, the composition comprising at least oneradiation-sensitive molecule and the CR-emitting radionuclide areadministered at the same time. For example, the composition comprisingat least one radiation-sensitive molecule and the radionuclide may beadministered as separate species. Alternatively, the compositioncomprising at least one radiation-sensitive molecule and theradionuclide may be administered as a probe as described in SectionI(b). The radionuclide can include those described in Section I(b). Thecomposition comprising at least one radiation-sensitive molecule caninclude those described in Section I(a).

In another embodiment, the composition comprising at least oneradiation-sensitive molecule and the CR-emitting radionuclide areadministered sequentially, wherein the composition comprising at leastone radiation-sensitive molecule may be administered first, followed byadministration of the CR-emitting radionuclide. For example, theCR-emitting radionuclide may be administered minutes, hours or daysafter administration of a composition comprising at least oneradiation-sensitive molecule. Accordingly, the CR-emitting radionuclidemay be administered from about 10 to about 15 minutes, or from about 15to about 30 minutes, or from about 30 minutes to about 45 minutes, orfrom about 45 minutes to 60 minutes after administration of acomposition comprising at least one radiation-sensitive molecule.Alternatively, the CR-emitting radionuclide may be administered fromabout 1 hour to about 2 hours, or from about 2 hours to about 3 hours,or from about 3 hours to about 4 hours, or from about 4 hours to about 5hours, or from about 5 hours to about 6 hours, or from about 6 hours toabout 7 hours, or from about 7 hours to about 8 hours afteradministration of a composition comprising at least oneradiation-sensitive molecule. In another embodiment, the CR-emittingradionuclide may be administered from about 1 day to about 2 days, orfrom about 2 days to about 3 days, or from about 3 days to about 4 days,or from about 4 days to about 5 days, or from about 5 days to about 6days, or from about 6 days to about 7 days after administration of acomposition comprising at least one radiation-sensitive molecule.

In still another embodiment, administration of the composition andadministration of the radionuclide may be repeated. For example,administration of the composition may be repeated, administration of theradionuclide may be repeated, or administration of both may be repeated.The repeating interval may be daily, bi-weekly, bi-monthly or monthly.In an embodiment, administration of the radionuclide is repeated. In anexemplary embodiment, administration of the radionuclide is repeated ondays 2 and 4 following administration of the composition.

(i) Composition

Suitable methods for administration of a composition comprising at leastone radiation-sensitive molecule include but are not limited to oral,intravenous, sublingual, intraperitoneal, subcutaneous, or intratumoraladministration. In an exemplary embodiment, intratumoral administrationis employed. In another exemplary embodiment, intravenous administrationis employed.

For therapeutic applications, a therapeutically effective amount of acomposition as described in Section I(a) is administered to a subject. A“therapeutically effective amount” is an amount of the compositionsufficient to produce a measurable biological tumor response (e.g., animmunostimulatory, an anti-angiogenic response, a cytotoxic response, ortumor regression) upon activation by a radionuclide. Actual dosagelevels of a composition can be varied so as to administer an amount ofthe composition that is effective to achieve the desired therapeuticresponse for a particular subject. The selected dosage level will dependupon a variety of factors including the properties of the compositionwhich may include the properties of the radiation-sensitive molecule,the combination of radiation-sensitive molecules, the properties of thetargeting agent, the properties of the radionuclide, the opticalproperties of the target tissue, formulation, route of administration,combination with other drugs or treatments, tumor size and longevity,and the physical condition and prior medical history of the subjectbeing treated. In one embodiment, a minimal dose is administered, anddose is escalated in the absence of dose-limiting toxicity.Determination and adjustment of a therapeutically effective dose, aswell as evaluation of when and how to make such adjustments, are knownto those of ordinary skill in the art of medicine. In one embodiment, atherapeutically effective amount of a composition for localizedapplication may be from about 0.5 μg/ml to about 50 μg/ml. In anotherembodiment, a therapeutically effective amount may be from about 1 μg/mlto about 15 μg/ml. In yet another embodiment, a therapeuticallyeffective amount may be less than 0.5 μg/ml. In still yet anotherembodiment, a therapeutically effective amount may be about 0.5 μg/ml,about 1 μg/ml, about 1.5 μg/ml, about 2 μg/ml, about 2.5 μg/ml, about 3μg/ml, about 3.5 μg/ml, about 4 μg/ml, about 4.5 μg/ml, about 5 μg/ml,about 5.5 μg/ml, about 6 μg/ml, about 6.5 μg/ml, about 7 μg/ml, about7.5 μg/ml, about 8 μg/ml, about 8.5 μg/ml, about 9 μg/ml, about 9.5μg/ml, about 10 μg/ml, about 11 μg/ml, about 12 μg/ml, about 13 μg/ml,about 14 μg/ml, about 15 μg/ml about 20 μg/ml, about 25 μg/ml, about 30μg/ml, about 35 μg/ml, about 40 μg/ml, about 45 μg/ml, or about 50μg/ml. Alternatively, a therapeutically effective amount may be lessthan about 0.5 μg/ml or greater than about 50 μg/ml. In an exemplaryembodiment, a therapeutically effective amount of a composition is 2.5μg/ml. For systemic administration, a therapeutically effective amountof a composition may be from about 0.1 mg/kg to about 50 mg/kg. Inanother embodiment, a therapeutically effective amount of a compositionmay be from about 0.1 mg/kg to about 10 mg/kg. In still anotherembodiment, a therapeutically effective amount of a composition may befrom about 0.5 mg/kg to about 1.5 mg/kg. For example, a therapeuticallyeffective amount may be be about 0.1 mg/kg, about 0.2 mg/kg, about 0.3mg/kg, about 0.4 mg/kg, about 0.5 mg/kg, about 0.6 mg/kg, about 0.7mg/kg, about 0.8 mg/kg, about 0.9 mg/kg, or about 1.0 mg/kg.Additionally, a therapeutically effective amount may be about 1.0 mg/kg,about 1.5 mg/kg, about 2.0 mg/kg, about 2.5 mg/kg, about 3.0 mg/kg,about 3.5 mg/kg, about 4.0 mg/kg, about 4.5 mg/kg, about 5.0 mg/kg,about 5.5 mg/kg, about 6.0 mg/kg, about 6.5 mg/kg, about 7.0 mg/kg,about 7.5 mg/kg, about 8.0 mg/kg, about 8.5 mg/kg, about 9.0 mg/kg,about 9.5 mg/kg, or about 10.0 mg/kg. Alternatively, a therapeuticallyeffective amount may be less than about 0.1 mg/kg or greater than about10 mg/kg.

For diagnostic applications, a detectable amount of a composition isadministered to a subject. A “detectable amount” is an amount of thecomposition sufficient to produce a detectable signal in vivo or invitro upon activation by a radionuclide. A “detectable signal” is asignal derived from a radioactive substance, radiation-sensitivemolecule, and the like. The detectable signal is detectable anddistinguishable from other background signals that are generated fromthe subject or sample. In other words, there is a measurable andstatistically significant difference (e.g., a statistically significantdifference is enough of a difference to distinguish among the detectablesignal and the background, such as about 0.1%, 1%, 3%, 5%, 10%, 15%,20%, 25%, 30%, or 40% or more difference between the detectable signaland the background) between detectable signal and the background.Standards and/or calibration curves can be used to determine therelative intensity of the detectable signal and/or the background. Adetectable amount will vary according to a variety of factors, includingbut not limited to the activity of the composition, formulation of thecomposition, the radiation-sensitive molecule, the combination ofradiosensitive-molecules, the route of administration, combination withother drugs or treatments, the size and longevity of the tumor orsuspected tumor, the physical condition and prior medical history of thesubject, the method of imaging and parameters related thereto,metabolism of the composition in the subject, the stability of thecomposition, and the time elapsed following administration of thecomposition prior to imaging. Thus, a detectable amount can vary and canbe tailored to a particular application. After study of the presentdisclosure, and in particular the Examples, it is within the skill ofone in the art to determine such a detectable amount. For localizedapplication, in one embodiment, a detectable amount of a composition toproduce a detectable signal in vivo may be from about 50 μg/ml to about1000 μg/ml. In another embodiment, a detectable amount may be greaterthan 1000 μg/ml. In yet another embodiment, a detectable amount may beless than 50 μg/ml. In still yet another embodiment a detectable amountmay be about 50 μg/ml, 100 μg/ml, about 150 μg/ml, about 200 μg/ml,about 250 μg/ml, about 300 μg/ml, about 350 μg/ml, about 400 μg/ml,about 450 μg/ml, about 500 μg/ml, about 550 μg/ml, about 600 μg/ml,about 650 μg/ml, about 700 μg/ml, about 750 μg/ml, about 800 μg/ml,about 850 μg/ml, about 900 μg/ml, about 950 μg/ml or about 1000 μg/ml.In an exemplary embodiment, a detectable amount of a composition is 250μg/ml. For systemic administration, a detectable amount of a compositionmay be from about 0.1 mg/kg to about 50 mg/kg. In another embodiment, atherapeutically effective amount of a composition may be from about 0.1mg/kg to about 10 mg/kg. In still another embodiment, a detectableamount of a composition may be from about 0.5 mg/kg to about 1.5 mg/kg.For example, a detectable amount may be be about 0.1 mg/kg, about 0.2mg/kg, about 0.3 mg/kg, about 0.4 mg/kg, about 0.5 mg/kg, about 0.6mg/kg, about 0.7 mg/kg, about 0.8 mg/kg, about 0.9 mg/kg, or about 1.0mg/kg. Additionally, a detectable amount may be about 1.0 mg/kg, about1.5 mg/kg, about 2.0 mg/kg, about 2.5 mg/kg, about 3.0 mg/kg, about 3.5mg/kg, about 4.0 mg/kg, about 4.5 mg/kg, about 5.0 mg/kg, about 5.5mg/kg, about 6.0 mg/kg, about 6.5 mg/kg, about 7.0 mg/kg, about 7.5mg/kg, about 8.0 mg/kg, about 8.5 mg/kg, about 9.0 mg/kg, about 9.5mg/kg, or about 10.0 mg/kg. Alternatively, a detectable amount may beless than about 0.1 mg/kg or greater than about 10 mg/kg.

(ii) Radionuclide

Suitable methods for administration of a radionuclide include but arenot limited to oral, intravenous, sublingual, intraperitoneal,subcutaneous, or intratumoral administration. In an exemplaryembodiment, intratumoral administration is employed. In anotherexemplary embodiment, intravenous administration is employed.

For therapeutic applications, a therapeutically effective amount of aradionuclide is administered to a subject. A “therapeutically effectiveamount” is an amount of the radionuclide sufficient to activate theradiation-sensitive molecule to produce a measurable biological tumorresponse (e.g., an immunostimulatory, an anti-angiogenic response, acytotoxic response, or tumor regression). Actual dosage levels of aradionuclide can be varied so as to administer an amount of theradionuclide that is effective to achieve the desired therapeuticresponse for a particular subject. The selected dosage level will dependupon a variety of factors including the properties of the radionuclide,the properties of the radiation-sensitive molecule(s), the opticalproperties of the target tissue, formulation, route of administration,combination with other drugs or treatments, tumor size and longevity,and the physical condition and prior medical history of the subjectbeing treated. In one embodiment, a minimal dose is administered, anddose is escalated in the absence of dose-limiting toxicity.Determination and adjustment of a therapeutically effective dose, aswell as evaluation of when and how to make such adjustments, are knownto those of ordinary skill in the art of medicine. In one embodiment, atherapeutically effective amount of a radionuclide may be from about 0.1mCi to about 2 mCi. In another embodiment, a therapeutically effectiveamount may be from about 0.1 mCi to about 1.5 mCi. In still anotherembodiment, a therapeutically effective amount may be from about 0.1 mCito about 0.5 mCi. In yet another embodiment, a therapeutically effectiveamount may be less than 0.1 mCi. In still yet another embodiment, atherapeutically effective amount may be about 0.1 mCi, about 0.15 mCi,about 0.2 mCi, about 0.25 mCi, about 0.3 mCi, about 0.35 mCi, about 0.4mCi, about 0.45 mCi, about 0.5 mCi, about 0.55 mCi, about 0.6 mCi, about0.65 mCi, about 0.7 mCi, about 0.75 mCi, about 0.8 mCi, about 0.85 mCi,about 0.9 mCi, about 0.95 mCi or about 1 mCi. Alternatively, atherapeutically effective amount may be about 1.0, about 1.1 mCi, about1.2 mCi, about 1.3 mCi, about 1.4 mCi, about 1.5 mCi, about 1.6 mCi,about 1.7 mCi, about 1.8 mCi, about 1.9 mCi, or about 2.0 mCi. In anexemplary embodiment, a therapeutically effective amount of aradionuclide is 0.25 mCi. In another exemplary embodiment, atherapeutically effective amount of a radionuclide is 1.0 mCi. In yetanother exemplary embodiment, a therapeutically effective amount of aradionuclide is 100-fold lower than the current paradigm in clinicalnuclear radiotherapy. A skilled artisan will understand that differentradionuclides may have different levels of standard dose used foradministration.

For diagnostic applications, a detectable amount of a radionuclide isadministered to a subject. A “detectable amount” is an amount of theradionuclide sufficient to activate the radiation-sensitive molecule toproduce a detectable signal in vivo or in vitro. A detectable amountwill vary according to a variety of factors, including but not limitedto the activity of the radionuclide, the route of administration,combination with other drugs or treatments, the size and longevity ofthe tumor or suspected tumor, the physical condition and prior medicalhistory of the subject, the method of imaging and parameters relatedthereto, metabolism of the radionuclide in the subject, the stability ofthe radionuclide, and the time elapsed following administration of theradionuclide prior to imaging. Thus, a detectable amount can vary andcan be tailored to a particular application. After study of the presentdisclosure, and in particular the Examples, it is within the skill ofone in the art to determine such a detectable amount. In one embodiment,a detectable amount of a radionuclide to activate a radiation-sensitivemolecule in vivo may be from about 0.1 mCi to about 1.5 mCi. In anotherembodiment, a detectable amount may be from about 0.1 mCi to about 0.5mCi. In yet another embodiment, a detectable amount may be less than 0.1mCi. In still yet another embodiment, a detectable amount may be about0.1 mCi, about 0.15 mCi, about 0.2 mCi, about 0.25 mCi, about 0.3 mCi,about 0.35 mCi, about 0.4 mCi, about 0.45 mCi, about 0.5 mCi, about 0.55mCi, about 0.6 mCi, about 0.65 mCi, about 0.7 mCi, about 0.75 mCi, about0.8 mCi, about 0.85 mCi, about 0.9 mCi, about 0.95 mCi or about 1 mCi.Alternatively, a detectable amount may be about 1.0, about 1.1 mCi,about 1.2 mCi, about 1.3 mCi, about 1.4 mCi, or about 1.5 mCi. In anexemplary embodiment, a detectable amount of a radionuclide is 0.25 mCi.In another exemplary embodiment, a detectable amount of a radionuclideis 1.0 mCi.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples that follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Introduction for Examples 1-6

Photodynamic therapy (PDT) has seen tremendous advancements in recentyears, with major focus on improving the tissue specificity ofphotosensitizers (PS) and tissue depth penetration of light. Theseefforts highlight the enormous potential of PDT as a viable treatmentregimen, but also expose the challenges that must be overcome to realizethe full benefits of this treatment paradigm. Regardless of themechanism of action, both Type I (direct transfer of radical ions froman activated PS to biomolecules) and Type II (direct generation ofsinglet oxygen species by an activated PS) PDT rely on reactive oxygenproduct for therapeutic effect′. This basic assumption implies that PDTwill be less efficient in hypoxic conditions such as those found in manysolid tumors². Therefore, an oxygen independent free radical generatingphotodynamic event could address this fundamental problem.

Biocompatible inorganic nanoparticles are attractive alternatives toconventional PS because of their large surface area, excellent payloadcapacity, and high reactivity^(3,4). Previous studies have shown thattitanium dioxide (TiO₂) nanoparticles are excellent photocatalysts thatcan absorb ultraviolet (UV) light (λ<385 nm) with high efficiency andgenerate free radicals such as hydroxyl and superoxide species throughelectron-hole pair production⁵. Generation of hydroxyl radicals throughelectron-hole transfer to chemisorbed H₂O is an oxygen-independentprocess, whereas superoxide radical generation requires aerated aqueousmedia for electron transfer to molecular oxygen⁶⁻⁹. Of these twoproducts, the highly cytotoxic hydroxyl radicals are the key speciesformed during the photocatalytic oxidation on the surface of TiO₂ inaqueous solvents^(7,9). These features have motivated the use of TiO₂ asa PS to induce cell death¹⁰⁻¹⁵. However, the shallow penetration of UVlight (<0.5 mm in tissue) has confined most of the previous studies toin vitro models of human diseases.

The limited penetrability of light in tissues remains the Achilles heelfor realizing the full potential of light based imaging and therapeutictechniques. Technological advances in bioluminescence imaging and lowlight detection techniques have prompted a fresh outlook at usingCerenkov radiation (CR) as a light source for molecular imaging¹⁶⁻²⁰. CRoccurs when charged particles such as positrons or electrons travelfaster than the speed of light in a given medium, emitting light rangingfrom 250-600 nm²¹. Positron emission tomography (PET) isotopes are anideal source for CR because of their high positron (13⁺) emission decayand their short half-life. 13⁺ particles travel short distances (<1 mm)in tissues, during which CR is first emitted before they undergoannihilation²². These data suggest that the broad CR luminescencespectrum and the availability of many clinical PET radionuclides couldovercome the difficulty of activating TiO₂ in the UV region for in vivoPDT. Therefore, we hypothesized that CR-mediated excitation of TiO₂nanoparticles will generate hydroxyl radicals for molecular oxygen- anddepth-independent PDT (CR-PDT). To test this hypothesis, we chose ⁶⁴Cuas the CR source because of its relatively short half-life (12.7 h),significant β decay (β⁺:19%, β⁻:39%), and availability at low cost.Using tumor cells and tumor-bearing mice, we demonstrate for the firsttime that the synergistic effect of low activity ⁶⁴Cu (≦0.25 mCi) andTiO₂ nanoparticles in tumors was sufficient to eradicate tumors in vivothrough CR-mediated depth-independent PDT. We also demonstrated that theintrinsic luminescence properties of TiO₂ nanoparticles can provideluminescence for optical imaging, allowing these materials toeffectively serve as a theranostic agent for depth-independentCR-mediated PDT and monitoring of treatment response by luminescenceimaging.

Example 1 Physical Characterization

TiO₂ typically exists in two tetragonal forms, anatase and rutile, whichdiffer in their crystal lattice structure²³. We employed anatase formfor CR-PDT studies because of its smaller size (<25 nm) and higherphotoactivity than the rutile form. The higher photoactivity of anataseis mainly due to the extent and nature of surface hydroxyl groups thatare generally associated with the surface of colloidal TiO₂ in water²⁴.In its native state, TiO₂ exhibits concentration dependent cytotoxicity,and unlike PDT, this toxicity cannot be controlled^(25,26). To eliminatethis undesirable effect and improve biocompatibility, we coated thenanoparticles separately with polyethylene glycol (PEG) and dextran(FIG. 1A-C). We synthesized TiO₂-PEG and TiO₂-Dextran conjugates byreacting TiO₂ with PEG (Molecular Weight: 400 Da) or dextran (MolecularWeight: 5,000 Da) in a sonicator to facilitate formation of Ti—O—Cbonds. These bonds are a result of a combination of molecular adsorptionand condensation reactions²⁷. Transmission electron microscope (TEM)analysis showed that these modifications transformed the TiO₂nanoparticle aggregates into monodisperse nanoparticles and smallnanoclusters for PEG and dextran coatings, respectively (FIG. 1D-F).Possibly, each dextran chain, which is significantly longer than PEG,interacted with multiple TiO₂ particles, creating a network of TiO₂nanoclusters. Solution phase characterization of nanoparticles suggestswell-defined dispersions and neutral charge densities after PEG anddextran coating (FIG. 1G-I and Table 1).

TABLE 1 Physico-chemical characterization of bare and coated TiO₂nanoparticles. Hydro- dynamic Poly- Zeta Diameter dispersity PotentialMobility Sample (nm) Index (mV) (μmcm/Vs) TiO₂ 454 ± 40 1.00 −17.1 ±4.6  −1.34 TiO₂-Dextran 110 ± 23 0.44 0.6 ± 3.7 0.04 (MW: 5,000)TiO₂-PEG 30 ± 6 0.26 4.3 ± 3.6 0.33 (MW: 400) The hydrodynamic size andzeta potential of the coated and uncoated TiO₂ nanoparticles inphosphate buffered saline (PBS) were measured using a Malvern Zetasizer.Hydrodynamic diameter was extracted by cumulant analysis of the data andPolydispersity index from cumulant fitting. Each value is the average ofthree experiments ± s.e.m.

Example 2 In Vitro CR-PDT

We assessed the biocompatibility of TiO₂, TiO₂-PEG, and TiO₂-dextrannanoparticle (FIG. 6). TiO₂ particles are known to induce apoptosis incells at concentrations exceeding 5 μg/ml through the caspase-8(initiator) to caspase-3 (effector) pathway²⁸. Hence, it is important todelineate intrinsic toxicity from CR-mediated phototoxicity.

We found that TiO₂ particles did not induce apoptosis at <4 μg/ml andthat ⁶⁴Cu was non-toxic at activity <0.25 mCi (FIG. 7). Therefore, weused these nontoxic doses of TiO₂ (2.5 μg/ml) and ⁶⁴Cu activity (<0.25mCi) to determine the effects of surface coating on PDT. Biologicallyinert PEG and dextran are known to prevent nonspecific uptake ofnanoparticles by cells²⁹. However, we observed that TiO₂ particlespermeated into cells, irrespective of surface coating or cell type. Thisindiscriminate cellular uptake suggests a nonspecific endocytosismechanism most likely through macropinocytosis, with “leaky” vesiclesthat are usually 500-2,000 nm in diameter³⁰. Most of the nanoparticleseventually localize in the lysosomes when the macropinosomes merge intolate endosomes and lysosomes (FIG. 8). When treated with ⁶⁴Cu, the cellviability of TiO₂-Dextran and TiO₂-PEG loaded cells were 52% and 24%,respectively, compared to the untreated cells (FIG. 2A). The enhancedPDT effect of TiO₂-PEG can be attributed to the nature of surfacecoating. Photocatalysis is a surface phenomenon that mediates the PDTeffect of TiO₂. Excessive adsorption of the polymers usually shields theparticles from absorbing incident UV light. This phenomenon can limitthe redox reaction that occurs on the surface and severely compromisethe efficiency of the hydroxyl radical generation process. In contrastto TiO₂-PEG, the relatively higher Molecular Weight (MW) of dextranprobably favored a denser surface coverage, a process that wouldconsiderably reduce the exposed TiO₂ surface area. This eventuallytranslates to lower photocatalytic potential and decreased PDT effect.

We further characterized TiO₂-PEG adducts to determine the lowestactivity of ⁶⁴Cu required to induce optimal PDT effect. We observedsignificant cell death at 0.1 mCi (FIG. 2B and FIG. 9). Quantitativeassays using Hydroxyphenyl fluorescein (HPF) for hydroxyl radicals andMitosox dye for superoxide radical confirmed relatively high levels ofboth the species at lower 0.25 mCi) than higher (>0.25 mCi)⁶⁴Cu (FIG. 2Cand FIG. 10). This result suggests that hydroxyl and superoxide radicalgeneration from TiO₂ is highest at 0.25 mCi, which is consistent with aprevious report that demonstrated maximum photocatalytic activity at lowUV light intensity, caused by recombination losses occurring at higherintensities³¹.

Hydroxyl radicals, which propagate within short distances (up to ˜3 nm)are extremely short lived species with an in vivo half-life of 10⁻⁹s^(32,33) They are highly reactive species, non-diffusible across cellmembranes, culminating into a highly pronounced local action³⁴. Incontrast, superoxide radicals are more stable species that can travelacross cell membranes with a long diffusion distance of ˜320 nm³⁵.Therefore, we evaluated the cytocidal effect of intracellular versusextracellular TiO₂ particles after treatment with ⁶⁴Cu (0.25 mCi). Weobserved that a majority of the cells (>95%) with extracellular TiO₂were viable (green), but cells loaded with intracellular TiO₂ weremostly necrotic (red) as shown in FIG. 2D,E. The minimal PDT effect oncells with extracellular TiO₂ strongly suggests that hydroxyl instead ofsuperoxide radicals are responsible for the cytotoxic effect. However,the PDT outcome is less dependent on the intracellular or extracellulardistribution profile of ⁶⁴Cu because the UV rays produced by CR arecapable of traversing cell membranes to activate the TiO₂ particles(FIG. 2F).

Example 3 TiO₂ Imaging

Photoluminescence of anatase TiO₂ as an indirect band gap semiconductoris well documented^(36,37). It is characterized by strong absorption inthe UV region (λ=274 nm), relatively strong luminescence in the visibleregion at 391 nm and 465 nm, and weak emissions at 749 nm (FIG. 3A).However, there are no reports on exploiting the photoluminescence ofTiO₂ for in vitro and in vivo optical imaging. In this study, weobserved luminescence in cells containing TiO₂ with concentrations aslow as 3 μg/ml (FIG. 3C and FIG. 11). This is consistent with ourspectroscopic studies where no marked change in the luminescence profileof cell internalized vs. cell-free TiO₂ nanoparticles (FIG. 3B) wasnoticed. The coating of TiO₂ by PEG also did not alter thenanoparticles' luminescence (FIG. 3C,D). The strong luminescence of TiO₂was used to determine the spatial distribution and localization of TiO₂within cells and tissue. Epifluorescence microscopy revealed the broadexcitability and imaging of intracellular TiO₂ using both FITC(Excitation/Emission: 490/545 nm) and Cy5 (Excitation/Emission: 630/710nm) filter sets (FIG. 3C). Fluorescence spectroscopic studies alsoconfirmed their excitability at 488 nm and 633 nm (FIG. 12).Interestingly, the cell labeling concentration of TiO₂ is within thelimit of conventional fluorescent nanoparticles (11 ng/ml-60 μg/ml),allowing the use of this approach for routine cell imaging studies.Confocal microscopy revealed distinctly identifiable crystallineluminescent particles, evenly distributed in the cytoplasm (FIG. 3E). Az-scan 3D reconstruction of the cell shows the particles well dispersedin the cytoplasm around the nucleus (FIG. 3F).

Consistent with the broad CR spectral range that tapers off from the UVto the far visible region (FIG. 4A), we observed that the luminescenceof different stoichiometric amounts of TiO₂ decreased with increasingwavelength (FIG. 4B). Since absorption and scattering by tissues willplay a major role in in vivo imaging of TiO₂ luminescence, the minimumdetectable concentration in vivo subcutaneous tumor mimics wasdetermined. We did not detect appreciable radiance below 250 μg/ml ofTiO₂ (FIG. 4C). The increase in detection threshold from 3 μg/ml of TiO₂in vitro to 250 μg/ml of TiO₂ in vivo can be ascribed to enhancedattenuation of visible light by endogenous absorbers and scatteringeffects of tissue. Substitution of CR with external excitation light didnot result in observable luminescence. Although TiO₂ nanoparticlespossess significant NIR luminescence, the result demonstrates that UVand visible light, which has limited penetration depth in tissue,dominates the photoactivity of TiO₂. Finally, we demonstrated thefeasibility of using TiO₂ luminescence to image solid tumors in vivofollowing CR excitation of the nanoparticles. Similar luminosity seen inthe tumor mimics was observed in the pancreatic tumor xenograftsinjected with 0.25 mCi of ⁶⁴Cu and 250 μg/ml of TiO₂ (FIG. 4D). ⁶⁴Cu wascompared to ^(99m)Tc, a pure γ emitter, to demonstrate that CR was theexcitation source for TiO₂ luminescence. The result shows that only ⁶⁴Cuwas able to induce luminescence in TiO₂ (FIG. 4E). This result isconsistent with the required minimum energy of 263 keV for a 13 particleto produce CR³⁸, which is not attained by the 140 keV ^(99m) Tc γemitter.

Example 4 In Vivo CR-PDT of Tumor Mimics

Motivated by the efficient in vitro PDT and appreciable luminescence ofTiO₂-PEG, we explored in vivo PDT and imaging studies usingsubcutaneously injected cancer cells to mimic tumor mass. Pancreaticcancers are known to be extremely refractory to chemotherapy because ofthe extensive extracellular stromal encapsulation of the tumor cells andthe low vascularity that impedes drug delivery into tumor cells³⁹.Therefore, we chose the aggressive pancreatic tumor cell line, BxPC-3,for this study. The cells were loaded with TiO₂-PEG and ⁶⁴Cu, as well ascontrol models that include BxPC-3 cells alone, TiO₂-PEG loaded cells,and ⁶⁴Cu-loaded cells. Tumor growth was inhibited in the mice treatedwith TiO₂-PEG and ⁶⁴Cu loaded cells up to 30 days post-treatment (FIG.5A). In contrast, tumor growth of up to 18±3 mm³ was observed in allthree controls within 14-16 days post-treatment. This result suggeststhat PDT mediated by the combined TiO₂-PEG and ⁶⁴Cu can inhibit theprogression of the initial tumor cells to form a tumor mass, therebyeradicating tumor survival. The rapid development of solid tumors fromthe seed cultures in the control mice indicates minimal dark toxicity ofthese individual components in vivo at the administered doses.

Example 5 In Vivo CR-PDT of Tumor Xenografts

The seed culture method described above demonstrates a strongsynergistic cytocidal PDT effect between TiO₂ and ⁶⁴Cu. This led us toexplore the translation of the findings to solid tumor xenografts usingBxPC-3 and HT1080 fibrosarcoma cell lines. Unlike BxPC-3 cells whichrapidly form solid tumors, HT1080 cells form unencapsulated tumors withrelatively extensive vascularity⁴⁰. These two distinct histopathologictumor types provide a unique platform to assess the efficacy ofTiO₂-⁶⁴Cu PDT in vivo. We observed shrinkage of the tumor volume (TV) bya remarkable 40±5% in HT1080 tumor bearing mice after 48 h (FIG. 5B,D).However, the mice with BxPC-3 tumors did not show any signs ofregression even after 10 days (FIG. 5C). At this point, a second dose ofthe TiO₂-⁶⁴Cu cocktail was administered. The mice were observed for atotal of 60 days without any measurable sign of BxPC-3 tumor regression.Nonetheless, the growth of treated BxPC-3 tumor masses progressed at aslower rate (TV at day 60=1050±150 mm³) compared to untreated controltumors (2570±500 mm³), extending mean survival time nearly two-fold(FIG. 5B). On the contrary, all treated mice with the HT1080 tumorsshowed complete regression by 45 days and did not require additionaltreatment to maintain this effect (FIG. 5B,D). Further monitoring of thePDT treated HT1080 tumor bearing mice for an additional 4 monthsdemonstrated the initial tumor regression translated into completeremission without significant loss in body weight. Expectedly, theuntreated HT1080 tumors did not regress.

Example 6 Histopathology

As seen in the mosaic image of the total tumor mass (FIG. 5E,F), theuntreated and treated BxPC-3 tumor architecture have similar features,with only one side of the tumor margin in the treated tumor showingsigns of necrosis and erosion of tumor capsule. Fluorescence image ofthe necrotic area shows localization of TiO₂-PEG at the site (FIG. 5G).On closer inspection of the cellular organization in the tumor tissue,clusters of cells are encapsulated by dense desmoplastic reactionconsisting of collagen, fibroblasts and other extracellular matrixproteins (FIG. 5H). In conjunction with hypovascularity of BxPC-3 tumor,this feature prevents the particles from interacting with theproliferating tumor cells. As demonstrated in the in vitro study,TiO₂-PEG trapped in the basement membrane matrix is unable to cause celldeath because internalization and localization in the cytoplasm are aprerequisite for an effective PDT response. Therefore, the unique tumorarchitecture of BxPC-3 pancreatic cancer plays a major role in the poorresponsiveness to CR activatable PDT using TiO₂ nanoparticles. Thisfinding suggests that the endemic resistance of pancreatic tumors totherapy could be overcome by co-administering nanotherapeutics withreagents that can disrupt desmoplasia. Interestingly, we observedextensive necrosis in the treated fibrosarcoma section at 72 hpost-injection, as evidenced by the large empty pockets throughout thetumor mass (FIG. 5J). In comparison, the untreated control tumor had thetypical herringbone appearance characteristic of fibrosarcomas (FIG.5I). The fluorescence image reveals TiO₂-PEG is internalized by cells,apparent by the diffuse and uniform luminescence (FIG. 5K), whichcontrasts with the TiO₂-PEG distribution in the extracellular matrix asaggregates in the treated pancreatic cancer. The unencapsulated andhypervascularized tumor architecture of fibrosarcoma (FIG. 5L) probablyfacilitated uninhibited diffusion across the tumor milieu and subsequentcellular internalization of TiO₂-PEG by the proliferating tumor cells,making PDT achievable.

In summary, the successful demonstration of tumor regression andremission in a fibrosarcoma model and extending mean survival in ahighly aggressive pancreatic tumor model, using ⁶⁴Cu as photon sourceand TiO₂ as oxygen-independent PS, marks an important event in theapplicability of CR for PDT. The short lived PET isotopes can thereforebe used as a light source for both superficial and deep tissuetheranostics. More importantly, lower doses of radioactivity than thecurrent paradigm in clinical nuclear imaging and radiotherapy⁴¹(100-fold) are sufficient to generate effective PDT, thus reducingradiotoxicity significantly. Although the activity of PET isotopes usedwas low, the concentration of TiO₂ for in vivo imaging is 100-foldhigher than that required for PDT in vitro. Replacement of ⁶⁴Cu as CRsource with other PET isotopes such as ⁹⁰Y and ⁸⁹Zr with higher CR couldimprove light delivery and increase the luminescence yield from TiO₂,thereby bridging the difference in TiO₂ concentration required forimaging and PDT applications. CR delimits PDT from the traditionalconstraints of external light beam excitation source, such as depth ofpenetration. The wide spectrum of CR, from UV to far visible, can alsobe exploited to systematically excite a range of PS. However, the lowfluence of CR will be challenging to excite organic PS such as FDAapproved porphyrins, due to their low molar absorption coefficients. Incontrast, the high surface area and dimension of nanoparticlessufficiently generates free radicals for PDT from the low fluence of CR.The CR-mediated PDT intratumoral injection approach disclosed in thiswork has direct potential clinical applications. For example,transarterial radioembolization⁴² has been used to treat hepatocellularcarcinoma. Therefore, direct intratumoral administration of TiO₂ can beused for regional management of chemotherapy refractory cancers andreduce systemic toxicity. To develop a versatile strategy for deeptissue CR-mediated PDT of both primary and metastatic cancer cells,future studies will focus on improving the selectivity ofnanoparticulate PS through a targeted approach, where both theradionuclide and targeting moieties are conjugated to TiO₂.

Methods for Examples 1-6 Synthesis of TiO₂-PEG and TiO₂-Dextran

Anatase TiO₂ (1 mg; Sigma Aldrich Co. St. Louis, Mo.) was suspended indeionized water (1 ml) and probe sonicated for 10 min before furtherprocessing. To a solution containing 1:1 PEG 400 (250 μl) and deionizedwater (250 μl), 100 μl of the sonicated TiO2 was added and sonicated foran additional 10 min at room temperature (RT). Similarly, Dextran fromLeuconostoc mesenteroides (0.5 mg; Sigma Aldrich Co.) was added todeionized water followed by the TiO₂ solution and sonicated for 10 minat RT. The TiO₂-PEG and TiO₂-Dextran adducts were then filtered using a0.22 μm membrane syringe filter to isolate the dispersed nanoparticles.

Cell Culture:

4T1, BxPC-3 and HT1080 cell lines (American Type CultureCollection-ATCC) were cultured under recommended standard conditions.4T1 and BxPC-3 were cultured in RPMI-1640 medium containing 10% fetalbovine serum (FBS), L-glutamine (2 mM), penicillin (100 units/rip, andstreptomycin (100 μg/ml), incubated at 37° C. in a humidified atmosphereof 5% CO₂ and 95% air. HT1080 were cultured in Dulbecco's ModifiedEagle's Medium under similar conditions.

In Vitro Cell Viability Assay:

MTS(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)assay, a calorimetric assay for assessing viability of cell culture, wasperformed using CellTiter 96® _(AQueous) Non-Radioactive CellProliferation Assay kit (Promega Co.) according to the manufacturer'sinstructions.

PhiPhiLux®G₂D₂(Oncolmmunin Inc.) with a DEVDGI amino acid sequence,having the following excitation and emission peaks: λ_(ex)=552 nm andλ_(em)=580 nm was used following the manufacturer's instructions. 5×10⁵BxPC-3 cells per well were grown in an 8 well chamber culture slide (BDBiosciences), incubated with 12.5 μg/ml, 5 μg/ml, 2.5 μg/ml and 1.25μg/ml of TiO₂. The 10 μM stock of PhiPhiLux® G₂D₂ was diluted with 1:1RPMI medium to prepare a 2× dilution working stock. 100 μl of the 5 μMsubstrate was added to the adherent cell monolayer after removing themedium and incubated at 37° C. in a humidified, 5% CO₂ atmosphere for 1h. The substrate was gently washed away using DPBS buffer two times andimaged through confocal microscopy. Propidium iodide and Live/Dead® cellstains (Life Technologies Inc.) were used according to themanufacturer's instructions.

In Cellulo Hydroxyl and Superoxide Radical Assay:

Hydroxyphenyl fluorescein (HPF) with an excitation and emissionwavelength of 490 nm and 515 nm, respectively (Life Technologies Inc.)was used according to the manufacturer's instructions. Briefly, the 5 mMstock was diluted 1,000× to 5 μM working stock in DPBS. The TiO₂-⁶⁴Cutreated BxPC-3 cells grown in 8 well culture slides were immersed in theHPF working stock 4 h post treatment. The cells were incubated for 1 hbefore the dye solution was washed away and replaced with fresh DPBS.The cells were imaged using confocal microscopy using the 488 nm Argonion laser with emission set to 500-600 nm. Similarly, Mitosox Red (LifeTechnologies Inc.) with an excitation and emission wavelength of 510 nmand 580 nm, was used to detect superoxide radicals using manufacturer'sinstructions.

Chelation of ⁶⁴Cu to DOTA:

A 1 mg/ml stock solution of DOTA(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) (MacrocyclicsInc.) was prepared in 50 mM ammonium acetate buffer equilibrated to pH5.5. 50 μl of DOTA stock was added to 450 μl of ammonium acetate bufferfollowed by 5 mCi of ⁶⁴Cu in 5 μl of Hydrochloric acid. The reactionmixture was incubated at 45° C. for 1 h in a shaker. Unchelated ⁶⁴Cu wasremoved from the chelated DOTA-⁶⁴Cu using a Waters HPLC purificationsystem. The flow rate was set to 1 ml/min. The solvents were A-0.1%Trifluoracetic acid (TFA) in water and B-0.1% TFA in Acetylnitrile.After 5 min hold at 5% B the gradient was programmed linearly to 100% Bat 40 min. The sample was collected for 2 min at 6 min time pointscorresponding to the peak in the radiometer and UV detector. The samplewas then dried in a rotary shaker to remove TFA and acetylnitrile, for 4h before resuspending in DPBS.

Characterization:

Transmission electron microscopy images were acquired using a FEI TecnaiSpirit Transmission Electron Microscope operating at an accelerationvoltage of 200 kV. Dynamic light scattering measurements were takenusing a Malvern Zetasizer Nano ZS instrument equipped with a 633 nmlaser. All sizes reported were based on intensity average. Fluorescenceimages were acquired using an Olympus BX51 epi-fluorescence microscope.Fluorescence/reflectance cell images were taken with a 40× objectiveusing the mercury lamp of the microscope as the excitation source andCy5 filter set with an excitation and emission range of 620±60 nm and700±75 nm, respectively. Confocal microscopy images were acquired usingan Olympus FV1000 confocal microscope. Fluorescence/reflectance cellimages were taken with a 60× objective using He:Ne 633 nm excitationlaser and emission range of dichroic mirrors set to 655-755 nm.

In Vivo Tumor Model:

Balb/c and Athymic nu/nu mice were purchased from Frederick CancerResearch and Development Center. All studies were conducted incompliance with Washington University Animal Welfare Committee'srequirements for the care and use of laboratory animals in research. The4T1 tumors were generated by subcutaneous injection of 4×10⁶ cells in100 μl of DPBS in Balb/c mice. Likewise, BxPC-3 and HT1080 tumors weregenerated by subcutaneous injection of 4×10⁶ cells in 100 μl of DPBS inAthymic nu/nu mice.

In Vivo Imaging

Matrigel™ (BD Biosciences) was thawed at 4° C. and added to an equalvolume of TiO₂-PEG solutions with the following stoichiometries—500μg/ml, 250 μg/ml, 120 μg/ml and 60 μg/ml and 0.25 mCi ⁶⁴Cu in each vial.After reformulation the final titrations of TiO₂-PEG was 500 μg/ml, 250μg/ml, 120 μg/ml, 60 μg/ml and 30 μg/ml, respectively. Balb/c mice(n=3×4) were injected with 100 μl of the formulation subcutaneously intheir flank region. Additionally, BxPC-3 tumor bearing Athymic nu/numice (n=3) were injected with 250 μg/ml of TiO₂ admixed with 0.25 mCi⁶⁴Cu in 50 μl of DPBS directly into the tumor. The mice were imagedusing the IVIS Lumina XR multimodal imaging system (PerkinElmer Inc.)immediately pi. Fluorescence imaging was performed using an excitationand emission wavelength of 640 nm and 700 nm, respectively, 60 sexposure with 2×2 binning. Luminescence images were acquired withLivinglmage software using a 695-770 nm emission filter, 3 min exposureand 4×4 binning. Region of interest (ROI) analyses were performed usingLivinglmage or ImageJ software. Luminescence intensity expressed asRadiance was recorded and normalized to controls. Statisticalsignificance was calculated using Graph Pad Prism software.

Photodynamic Therapy: CR-PDT of Tumor Mimic:

BxPC-3 cells were treated with 2.5 μg/ml of TiO₂-PEG and incubatedovernight to facilitate internalization. The cells were centrifuged at3500 rpm for 5 min and resuspended in DPBS to get rid ofnon-internalized TiO₂-PEG. This cycle was repeated three times. 8×10⁶cells in 50 μl of DPBS were suspended in an equal volume of Matrigel™along with 0.25 mCi of ⁶⁴Cu. The gel was injected subcutaneously in theflank region of Athymic nu/nu mice (n=6). Three control groups, TiO₂-PEGloaded BxPC-3 cells in Matrigel™ (n=6), 0.25 mCi of ⁶⁴Cu in Matrigel™(n=6), and BxPC-3 cells in Matrigel™ (n=3), were also similarly injectedinto the mice. The animals were monitored for 30 days. The growingtumors were measured with calipers every two days and tumor volumecalculated using the equation: TV=(length×width²)/2. The TV was plottedversus time to analyze PDT effect on the seed culture.

CR-PDT of Solid Tumors:

BxPC-3 tumors in Athymic nu/nu mice (n=4) were injected with 2.5 μg/mlTiO₂-PEG and 0.25 mCi ⁶⁴Cu cocktail in 50 μl of DPBS. Two diametricallyopposite injection sites were chosen and 25 μl of the cocktail wasdelivered at each site. An untreated group (n=4) served as control.HT1080 tumors in Athymic nu/nu mice (n=4) were also treated similarly.Three groups, TiO₂-PEG treated mice (n=4), ⁶⁴Cu treated mice (n=4), anduntreated mice (n=4), served as controls. The mice were monitored for 60days with tumor volume measurements taken every two days using calipers.The weight and any physical signs for distress were also monitoredclosely. The tumor volume calculation and analysis of PDT effect onsolid tumors was conducted as described above. The mice with regressingtumors were monitored for an additional four months to determine whetherthe cancer was in remission.

Histology:

The BxPC-3 tumor bearing mice in the treatment and control groups weresacrificed sixty days after injection of TiO₂-⁶⁴Cu cocktail. Likewise,for HT1080 tumor bearing mice, the mice were sacrificed three days afterinjection of TiO₂-⁶⁴Cu cocktail. The tumors were harvested andsnap-frozen in OCT media for routine staining with hematoxylin and eosin(H&E). 10 μm tumor sections were made and imaged using epi-fluorescencemicroscopy at 4× and 20× magnification. Brightfield images of H&Estained sections at 4× were stitched together to generate a compositeimage of the entire tumor volume using MicroSuite software. Fluorescenceimages were taken at 20× magnification using Cy5 filter set.

References for Examples 1-6

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Introduction for Examples 7-10

The combination of light and photosensitizers offers a high degree ofcontrol for selective treatment of human diseases, eradication ofcontagious microbes, and understanding the molecular basis of drugresistance^(t2). Despite the promise of phototherapeutic interventions,such as photodynamic therapy (PDT), the shallow penetration of light intissue, use of high light power to activate photosensitizers, andreliance on tissue oxygenation to generate cytotoxic radicals confinePDT to superficial or endoscope accessible lesions′. Here, we report atwo-prong approach that uses tissue depth independent Cerenkov radiation(CR) from radionuclides (¹⁸F or ⁶⁴Cu), as well as low radiance and anoxygen independent photocatalyst, titanium dioxide (TiO₂)nanophotosensitizer (NPS), for CR-induced therapy (CRIT). We demonstratethat administration of tumor-targeted transferrin coated TiO₂ andradionuclides in tumor-bearing mice remarkably increased median survivalor achieved complete tumor remission. This work reveals a new paradigmfor harnessing low radiance-sensitive NPS to achieve efficientdepth-independent CR-mediated therapy.

Breakthroughs in light-based diagnostic and therapeutic interventionshave transformed medicine and biology, as evidenced by recent advancesin multiphoton microscopy, photoacoustic technology, targetedphotoablation of tissue, photothermal therapies, PDT, and image guidedsurgeries. Regardless of the method employed, light-based interventionssuffer from the rapid attenuation of light in tissue, confiningphototherapy to superficial lesions³, unless a fiber light source⁴ isused to access deep organs.

CR, a broad spectrum light (250-600 nm) produced by many clinical graderadionuclides⁵, could serve as a depth-independent light source forphoto-induced therapy. Several studies have demonstrated the use of CRfor molecular imaging⁶⁻⁸, but the low radiance of CR is less effectivein activating conventional photosensitizers used in PDT. In this work,we demonstrate that a photocatalyst, TiO₂ NPS, can efficiently harvestthe predominant UV light from CR for a therapeutic effect. Unlike someconventional photosensitizers which largely rely on tissue molecularoxygen to generate cytotoxic reactive oxygen species⁹, theoxygen-independent radical generation by TiO₂ NPS potentially extendsPDT to the treatment of hypoxic lesions, such as solid tumors¹⁰. Byincorporating a photoinitiator into the NPS, we demonstrate that CRIT isan effective therapeutic paradigm using low light power and lowconcentrations of NPS.

Example 7 Titanium Dioxide and Titanocene Photoagents for CRIT

TiO₂ NPS is a regenerative photocatalyst that produces predominantlyhydroxyl radicals (FIG. 13A) through electron-hole transfer tochemisorbed H₂O in an oxygen-independent process¹¹⁻¹³. Because of theirlarge surface area for efficiently harvesting UV light¹⁴, where CRquantum efficiency is highest¹⁵, and their ability to generate freeradicals at low CR radiance for localized cytotoxicityl⁴, we exploredthe use of TiO₂ NPS for CRIT. Three types of stable TiO₂ NPS weresynthesized for this study. The first, TiO₂-PEG NPS, was prepared byultrasonicating TiO₂ with PEG (Molecular Weight: 400 Da), whichtransformed the TiO₂ nanoparticle aggregates into small nanoclusters(FIG. 13C and Table 2)¹⁶. Due to the nonspecific distribution of thisNPS, it was used to determine tumor response to CRIT via anintra-tumoral administration route.

The second NPS was designed for intravenous injection (i.v.)administration. Because of the high demand for iron by rapidlyproliferating cells, many tumors overexpress transferrin (Tf)receptors¹⁷. We discovered that treatment of TiO₂ nanoclusters (FIG.13C) with Tf produces monodispersed TiO₂ NPS (FIG. 13C). Under neutralpH, sonication of high concentrations of Tf facilitates adsorption ofthe negatively charged Tf (isoelectric point=5.5) onto TiO₂ (isoelectricpoint=5.8), which stabilizes the monodispersed NPS throughprotein-protein electrostatic repulsions. Although bovine serum albuminwas previously used to prepare stable suspensions of TiO₂ nanoclustersin protein rich media¹⁸, our new method allows Tf to servesimultaneously as a TiO₂ monodispersant, stabilizer and a tumortargeting moiety.

An inherent flaw of receptor-mediated targeting strategies is that lowconcentrations of materials are delivered to the target tissue. Fortherapy, this could lead to suboptimal effects. To overcome thischallenge, we prepared the third NPS by incorporating titanocene (Tc)into the tumor-targeted NPS to amplify the therapeutic effect of CRIT atlow NPS concentrations in tissue. Tc is a photoinitiator that can beactivated by UV light to generate free radicals (FIG. 13B) throughphotofragmentation¹⁹. The cyclopentadienyl and titanium-centeredradicals from Tc fragmentation are generated in an oxygen independentfashion²⁰. Similar to TiO₂ (A_(max) 274 nm), the excitation energy forTc (A_(max) 250 nm) is in the UV spectrum (FIG. 17)^(14,19), whichfavors CRIT. Importantly, apo-Tf (Tf devoid of iron) binds Tc with highaffinity at the iron-chelating epitope²¹, such that Tc—TiO₂-Tf can besynthesized by simply adding Tc to a solution of TiO₂-Tf (FIG. 13C).

TEM and DLS analyses show monodispersed (polydispersity index=0.08)TiO₂-Tf and TiO₂-Tf-Tc nanoparticles with an average size distributionof 18±3 nm and a hydrodynamic diameter of 106±18 nm, respectively (Table2). Using the strong luminescence of TiO₂ in the visible region (FIG.17D), we observed CR-mediated TiO₂ luminescence in ⁶⁴Cu (αβ particleemitter) treated samples, but not in ^(99m)Tc (a pure γ emitter) treatedsamples, demonstrating that CR was the excitation source for TiO₂luminescence (FIG. 17E). This finding is supported by a previous studythat shows CR from ³²P can excite TiO₂ and cleave DNA²² in similarmanner as activation with white light²³.

TABLE 2 Physico-chemical characterization of TiO₂-PEG, TiO₂-Tf andTiO₂-Tf-Tc constructs. Hydro- dynamic Poly- Zeta Diameter dispersityPotential Mobility Sample (nm) Index (mV) (μmcm/Vs) TiO₂ 454 ± 40 1.00−17.1 ± 4.6 −1.34 TiO₂-PEG 268 ± 26 0.23  4.28 ± 1.9 0.33 TiO₂-Tf 106 ±18 0.08 −7.77 ± 3.7 −0.61 TiO₂-Tf-Tc 108 ± 13 0.09 −7.36 ± 3.6 −0.57 Thehydrodynamic size and zeta potential of the TiO₂ based constructs inphosphate buffered saline (PBS) were measured using a Malvern Zetasizer.Hydrodynamic diameter was extracted by cumulant analysis of the data andpolydispersity index from cumulant fitting. Each value is the average ofthree experiments ± s.e.m.

Example 8 Cellular Uptake of Photoagents and In Vitro CRIT Assessment

Using an electron microscope (EM), we demonstrated sub-cellularlocalization of the TiO₂-Tf NPs in the endo-lysosomal compartment ofHT1080 tumor cells. NPS-free Tf successfully inhibited the endocytosis,suggesting internalization is mediated by the Tf receptor (FIG. 14A).TiO₂ NPS and Tc are known to induce apoptosis in cells at concentrations≧5 μg/ml and 12.5 μg/ml, respectively^(24,25). To delineate theintrinsic from CR-mediated toxicity, we used TiO₂-Tf, Tc-Tf, andTiO₂-Tf-Tc NPS, as well as two radionuclides, ¹⁸F and ⁶⁴Cu. With ahalf-life of 1.83 h and predominantly 13 decay (β⁺: 97%), the widelyused PET imaging agent 2′-deoxy-2′-(¹⁸F)fluoro-D-glucose (FDG) issuitable for systemic administration, where its high specific activityand tumor-targeting capability²⁶ combine to deliver rapid and localizedCR for CRIT without prolonged exposure of healthy tissue toradioactivity. For intra-tumoral injection, where rapid regression oftumor growth is needed, we used ⁶⁴Cu, which has a half-life of 12.7 hand significant β decay (β⁺:19%, β⁻:39%). Our cytotoxicity analysisshowed that cell viability in 64Cu (<0.5 mCi (18.5 MBq)/0.1 ml) and FDG(1 mCi (37 MBq)/0.1 ml) treated cells was >95% relative to untreatedcontrols (FIG. 7B). Similarly, TiO₂ NPS or Tc did not induce apoptosisat <4 μg/ml and <10 μg/ml, respectively (FIG. 7A). Therefore, we useddoses below the toxicity threshold to determine efficacy of CRIT invitro.

When treated with FDG and ⁶⁴Cu, the viability of tumor cells preloadedwith NPS significantly decreased (FIG. 14B), suggesting low metabolicactivity and attenuated proliferation. Cellular analysis with thealkaline Comet Assay show various degrees of DNA mobility outside thenucleus (FIG. 14C) and that a significant percentage of treated cellsexhibited DNA damage, which correlated with the viability studies (FIG.14D). Compared to untreated cells (FIG. 14E), EM images of cells loadedwith TiO₂-Tf after treatment with FDG revealed features associated withboth necrosis, such as loss of cell membrane integrity and a vacuolatedcytosol (FIG. 14F), and apoptosis, such as dense nuclei, chromatinmargination, and excessive surface blebbing (FIG. 14G). However, cellstreated with Tc-Tf and FDG exhibited predominantly apoptotic features(FIG. 14H). Propidium iodide staining demonstrated oncotic cells withhigh uptake (FIG. 14I, middle) and low uptake (FIG. 14I, bottom) of thestain in TiO₂-Tf+FDG and Tc-Tf+FDG treated cells, respectively (seeTiO2-PEG+⁶⁴Cu related information in FIG. 9 and FIG. 10). Loss of cellmembrane integrity and disruption of the signal transduction pathwayleading to apoptosis and cell death is a hallmark of oxidative celldamage mediated by peroxyl, hydroxyl, and superoxide radicals via lipidperoxidation of the cell membrane²⁷. Peroxyl, cyclopentadienyl andmetal-centered radicals are known to be less disruptive than hydroxylradicals²⁸, which is consistent with the observation of oncotic cellswith lightly stained nuclei, indicating a lower degree of cellulardamage and that the cells are in late stages of apoptosis (FIG. 14J).

Using dyes that are sensitive to hydroxyl and peroxyl radicals(hydroxyphenyl fluorescein, HPF)²⁹, and superoxide radicals (Mitosox),we demonstrated that cells treated with TiO₂-Tf and FDG exhibited highlevels of both HPF and mitosox fluorescence (FIG. 14K). These resultssuggest the involvement of both hydroxyl and superoxide species in CRIT.Hydroxyl radicals are a highly reactive species and non-diffusibleacross cell membranes, culminating in a highly pronounced localaction³⁰. In contrast, superoxide radicals are a more stable speciesthat can travel across cell membranes with a long diffusion distance of˜320 nm³¹. Because CR can traverse cell membranes to activateintracellular and extracellular TiO₂ NPS, we used differences in thepropagation kinetics of the two radicals to delineate the contributionsof each radical species to CRIT. FIG. 14L shows that a majority of thecells (>95%) treated with extracellular TiO₂ and ⁶⁴Cu (0.5 mCi/0.1 ml)were viable, suggesting that the hydroxyl, and not the superoxide,radicals play a major role in CRIT. Although the interaction of betaparticles or gamma rays with TiO₂ NPS can mediate CRIT, this effect wasnot observed under our experimental conditions. We did not observe CRITafter treatment of TiO₂ NPS with the pure gamma emitter (^(99m)Tc) orafter FDG treatment of gold nanoparticles, which are radiosensitizersknown to generate photoelectrons and auger electrons upon interactingwith ionizing radiation and X-rays³² (FIG. 18).

Example 9 CRIT Through Intratumoral Administration of TiO₂ and ⁶⁴Cu

To demonstrate the in vivo application of CRIT, we administered a singlesub-cytotoxic dose of TiO₂-PEG NPS (2.5 μg/ml) and ⁶⁴Cu (0.5 mCi/0.1 ml)into mice bearing the aggressive HT1080 tumor model. We observed aremarkable shrinkage in tumor volume (40±5%) within 3 days of CRITinitiation (FIG. 15A,B). Complete tumor regression was achieved by 30days (FIG. 15B), translating into complete remission without significantloss in body weight up to 4 months post treatment. In contrast, theuntreated HT1080 tumors grew rapidly and the mice were euthanized by day15. Histologic analysis showed that untreated tumors had the typicalherringbone appearance characteristic of fibrosarcomas (FIG. 15C), buttreated tumors revealed extensive necrosis at 72 h post-injection, asevidenced by the large denuded areas throughout the tumor mass (FIG.15D). Although intra-tumoral administration of drugs is a viableadjuvant therapy for a variety of tumors such as liver (radionuclidetherapy through chemoembolization) or brain cancer, we expanded thepotential application of the system to i.v.-based CRIT.

Example 10 In Vivo Distribution and CRIT Through SystemicallyAdministered Photoagents and FDG

Labeling Tf with the dye Alexa 680 allowed us to determine the in vivodistribution and tumor uptake of the NPS. For TiO₂-Tf, the uptake washighest in tumors (FIG. 16A) relative to other organs, an outcome thatis rare for most nanoparticles (see FIG. 19 for additional information).The tumor to muscle ratio for TiO₂-Tf (9.5) is higher than that of Tfalone (5.3), which could be attributed to additional uptake due toenhanced permeability and retention effect. Inspired by this result, weintravenously administered a one-time dose of each TiO₂-Tf, Tc-Tf orTiO₂-Tf-Tc (1 mg/kg body weight) in different mice, followed by twodoses of FDG (0.87 mCi (32.19 MBq)/0.1 ml) within 72 h. The animals weremonitored over 45 days (FIG. 16B). We observed that the tumor growthrate for mice undergoing CRIT was considerably slower than in untreatedor other control mice. The average tumor volume for mice treated withTiO₂-Tf or Tc-Tf and FDG was four-fold smaller than the correspondingcontrols at day 15, when the control groups had to be euthanized beforethe tumors attained 2 cm limit imposed by our protocol. Importantly,mice treated with TiO₂-Tf-Tc and FDG showed superior response to CRIT,with an eight-fold smaller average tumor volume compared to the controlgroups. Median survival increased from 15±2 d, for the untreated andcontrol groups, to 30.5 d for TiO₂-Tf+FDG, 31 d for Tc-Tf+FDG, and aremarkable 50 d for TiO₂-Tf-Tc+FDG (FIG. 16C) treated mice. This resultsuggests a complementary effect of TiO₂ and Tc in the presence of FDG,leading to an additive effect in growth inhibition. We also observed theattenuation of tumor growth and a significant increase in mediansurvival to 21 d for TiO₂-Tf+FDG, 22 d for Tc-Tf+FDG, and 29 d forTiO₂-Tf-Tc+FDG, when mice were treated with lower dose of FDG (0.43 mCi(15.91 MBq)/0.1 ml) (FIG. 16D). However, administration of trace amountsof FDG (0.14 mCi (5.18 MBq)/0.1 ml activity) did not induce CRIT (FIG.16D). Taken together, the systemic and intra-tumoral CRIT data suggest apositive correlation between in vivo cell death and the intensity of CR.Clearly other factors such as duration of exposure, administered dose,and the type of radionuclide used will influence CRIT outcomes.

The high specific activity of FDG provided excellent images of tumorsbefore and after CRIT using positron emission tomography (PET). At tracelevels, FDG serves as an imaging agent without inducing CRIT (FIG.16E,F). After increasing the injected dose to >0.4 mCi (15.91 MBq)/0.1ml per mouse to trigger CRIT, FDG-PET imaging clearly demonstrates aremarkable decrease in FDG uptake in tumors that responded to CRIT.Image analysis shows selective destruction of proliferating cells in thetumor region, with one tumor revealing necrotic centers (FIG. 16E,F).Histological analysis of tumor sections of TiO₂-Tf and TiO₂-Tf-Tctreated mice reveals pronounced necrotic zones occupying approximately30% and 40% of the tumor mass, respectively (FIG. 16G). A significantlyhigh number of tumor infiltrating lymphocytes (TIL), primarilyneutrophils, and macrophages were observed among the necrotic cells.Large areas of the tumor exhibited loss of cellular architecture,probably due to scavenging of the necrotic debris by macrophages, asevidenced by large denuded pockets. However, in Tc-Tf and FDG treatedtumors, only 15% of the tumor mass was necrotic, with a high TILpopulation and a significantly higher distribution of apoptotic foci.These findings suggest that in addition to free radical mediated directdamage to cells, activation of the immune system against the tumor cellstriggered neutrophil and macrophage recruitment. As observed in our invitro studies, it appears that necrosis is the dominant feature of TiO₂based CRIT, while apoptosis mediated cell death when the Tc basedconstructs were used. The extended median survival in TiO₂-Tf-Tc treatedmice resulted from the additive bimodal cell death mechanism through thecombined effects of different radicals generated by the photocatalystand photoinitiator.

Off-target toxicity is a concern for i.v. based CRIT. This isparticularly important in the liver and kidneys, which are the mainelimination route for the materials. These organs are also sensitiveindicators of systemic toxicity caused by therapeutic interventions.Histological analysis of the liver and kidneys following CRIT did notshow any significant lesions in the organs, indicating CRIT wasselective for proliferating tumor cells (FIG. 20).

Conclusions For Examples 7-10

In this study, we have demonstrated a new approach for the use of lowradiance CR for phototherapy by designing NPS that are susceptible tosub-therapeutic doses of radioactivity. Additive effects ofcomplementary radical generation mechanisms of photocatalysts (hydroxylradicals) and photoinitiators (photofragmentation) enabled effectiveCRIT using tumor targeted NPS, where the tumor concentration isconventionally low. The astute use of Tf as a tumor-targeting agent a Tcchaperone, a TiO₂ chelator, a linker, and a dispersant to preventnanoparticle aggregation, ushers in a modular approach for NPS designand efficient tumor-targeted CRIT in vivo. Optimization of the dosingregimen could lead to complete tumor remission using i.v. administrationof the agents. Because of the established biocompatibility of allcomponents used in the study, our work creates a clear path to humantranslation. Although we focused on tumor therapy, the approachdescribed in this study is versatile, opening the possibility oftreating a variety of lesions in a depth- and oxygen-independent manner,thus overcoming the Achilles heel of phototherapeutic interventions.

Methods for Example 7-10

Synthesis of TiO₂-PEG TiO₂-Tf, Tc-Tf and TiO₂-Tf-Tc. Anatase TiO₂ (1 mg;Sigma Aldrich Co.) was suspended in deionized water (1 ml) to prepare aworking stock solution. PEG 400 (100 μl) was added to the TiO₂ solutionand sonicated using a probe sonicator for 10 min at room temperature(RT). The mixture was then dialyzed overnight against Dulbecco'sPhosphate Buffered Saline (DPBS) using a 3000 Da molecular weight cutoff(MWCO) Slide-A-Lyzer MINI Dialysis Device (Thermo Fisher ScientificInc.) to remove excess PEG. Working stock solutions of Tf were preparedby dissolving 5 mg of human apo-Tf (Sigma Aldrich Co.) in 1 ml DPBS, pH7.4. To prepare TiO₂-Tf, a 1:1 (v/v) solution of TiO₂ and Tf was mixedand probe sonicated in continuous mode for 5 min. The solution was thenimmediately passed through a 0.45 μm syringe filter to isolatemonodisperse nanoparticles. To prepare Tc-Tf, five-fold molar excess ofTc (Sigma Aldrich Co.) was added to human apo-Tf and incubated in ashaker for 2 h at room temperature (RT). A working stock of Tc wasinitially prepared in DMSO due to the low solubility of Tc in water andaqueous buffers. The mixture was then dialyzed overnight against DPBSusing a 3000 Da molecular weight cutoff (MWCO) Slide-A-Lyzer MINIDialysis Device to remove excess Tc. TiO₂-Tf-Tc was similarly preparedby incubating Tc with TiO₂-Tf conjugates and thereafter dialyzing toremove excess Tc.

Physicochemical Characterization.

Transmission electron microscopy images were acquired using a FEI TecnaiSpirit Transmission Electron Microscope (FEI) operating at anacceleration voltage of 200 kV. Dynamic light scattering measurementswere taken using a Malvern Zetasizer Nano ZS (Malvern Instruments Ltd.)instrument equipped with a 633 nm laser. Three measurements wereconducted for each sample with at least 10 runs, each run lasting 10 s.All sizes reported were based on intensity average. Absorption spectraof TiO2 and Tc were recorded on a Beckman Coulter DU 640 UV-visiblespectrophotometer (Beckman Coulter Inc.) and analyzed using GraphpadPrism statistical software. Fluorescence spectra of TiO₂ were recordedon a Fluorolog-3 spectrofluorometer (Jobin Yvon Horiba). The sample wasplaced in a quartz cuvette and measurements recorded in triplicates.

Cell Culture.

HT1080 fibrosarcoma cells (American Type Culture Collection-ATCC) werecultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10%fetal bovine serum (FBS), L-glutamine (2 mM), penicillin (100 units/rip,and streptomycin (100 μg/ml) at 37° C. in a humidified atmosphere of 5%CO₂ and 95% air. For cytotoxicity studies, concentration of 2.5 μg/ml ofthe TiO₂-Tf, Tc-Tf, and TiO₂-Tf-Tc NPS as well as 0.5 mCi/0.1 ml of FDGand ⁶⁴Cu were used.

TEM Analysis of Cells with TiO₂-77 and Tc-Tf.

For ultrastructural analysis, cells were fixed in 2%paraformaldehyde/2.5% glutaraldehyde (Polysciences Inc.) in 100 mMcacodylate buffer, pH 7.2 for 1 h at room temperature. Samples werewashed in cacodylate buffer and postfixed in 1% osmium tetroxide(Polysciences Inc.) for 1 h. Samples were then rinsed extensively indistilled water prior to en bloc staining with 1% aqueous uranyl acetate(Ted Pella Inc.) for 1 h. Following several rinses in water, sampleswere dehydrated in a graded series of ethanol and embedded in Eponate 12resin (Ted Pella Inc.). Sections of 95 nm were cut with a Leica UltracutUCT ultramicrotome (Leica Microsystems Inc.), stained with uranylacetate and lead citrate, and viewed on a JEOL 1200 EX transmissionelectron microscope (JEOL USA Inc.) equipped with an AMT 8 megapixeldigital camera (Advanced Microscopy Techniques).

In Vitro Cell Viability Assays.

The MTS(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)assay, a colorimetric assay for assessing cell viability was performedusing the CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assaykit (Promega Co.) according to the manufacturer's instructions. Thecells were incubated with the constructs and FDG for 48 h beforeanalysis.

The alkaline Comet Assay (Cell Biolabs Inc.) was performed using themanufacturer's protocol. Briefly, treated and untreated control cellswere removed from the flask by scraping. The cell suspension wascentrifuged and washed with ice-cold DPBS two times and resuspended at1×10⁵ cells/ml in ice-cold DPBS. Cells were embedded in low melt Cometagarose and plated on provided microscope slides. The cells were thenlysed with lysis buffer and treated with alkaline solution. The slideswere electrophoresed in alkaline solution at 1 Volt/cm with a setting of300 mAmp for 30 minutes. The slides were stained with Vista Green DNAdye after washing and drying. Fluorescence images were acquired using anOlympus BX51 epifluorescence microscope equipped with a CCD camera. %Tail DNA was estimated using the OpenComet (v1.3) plugin for Image Jsoftware.

Propidium iodide stain (Life Technologies Inc.) was used according tothe manufacturer's instructions. Fluorescence/reflectance cell imageswere taken with a 40× objective using the mercury lamp of the microscopeas the excitation source. FITC and Cy5 filter sets with anexcitation/emission range of 480±40/535±50 nm and 620±60/700±75 nm,respectively, were used. Confocal microscopy images were acquired usingan Olympus FV1000 confocal microscope. Fluorescence/reflectance cellimages were taken with a 60× objective using He:Ne 488 and 633 nmexcitation lasers and an emission range of dichroic mirrors set to455-575 nm and 655-755 nm, respectively. Fluorescence and reflectanceimage overlay with false color was performed using Fluoview FV10-ASWsoftware from Olympus (Center Valley, Pa.).

In Cellulo Hydroxyl and Superoxide Radical Assay.

Hydroxyphenyl fluorescein (HPF) with an excitation and emissionwavelength of 490 nm and 515 nm, respectively (Life Technologies Inc.)was used according to the manufacturer's instructions. Briefly, the 5 mMstock was diluted to a 5 μM working stock in DPBS. Cells were grown in 8well slides. The TiO₂-Tf, Tc-Tf and TiO₂-Tf-Tc and FDG treated HT1080cells were immersed in the HPF working stock 4 h post treatment. Thecells were incubated for 1 h before the dye solution was washed away andreplaced with fresh DPBS. The cells were imaged using confocalmicroscopy using the 488 nm Argon ion laser with emission set to 500-600nm. Similarly, Mitosox Red (Life Technologies Inc.) with an excitationand emission wavelength of 510 nm and 580 nm, was used to detectsuperoxide radicals, following the manufacturer's instructions.

Chelation of ⁶⁴Cu to DOTA.

For experiments with ⁶⁴Cu, typically chelation is essential to mitigatetoxicity from Cu(II) ions. A 1 mg/ml stock solution of DOTA(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) (MacrocyclicsInc.) was prepared in 50 mM ammonium acetate buffer equilibrated to pH5.5. 50 μl of DOTA stock was added to 450 μl of ammonium acetate bufferfollowed by 5 mCi (185 MBq) of ⁶⁴Cu in 5 μl of hydrochloric acid. Thereaction mixture was incubated at 45° C. for 1 h in a shaker.Non-chelated ⁶⁴Cu was removed from the chelated DOTA-⁶⁴Cu using a WatersHPLC purification system. The flow rate was set to 1 ml/min. Thesolvents were A-0.1% Trifluoracetic acid (TFA) in water and B-0.1% TFAin acetonitrile. After 5 min hold at 5% B the gradient was programmedlinearly to 100% B at 40 min. The sample was collected for 2 min at 6min time points corresponding to the peaks in the radiometer and UVdetector. The sample was then dried in a rotary shaker to remove TFA andacetonitrile for 4 h before re-suspending in DPBS.

Matrigel Based Cell Studies.

Matrigel™ (BD Biosciences) was thawed at 4° C., added to an equal volumeof TiO2 solution, and plated on 8 well chamber slides. HT1080 cells weregrown on the plated slides before ⁶⁴Cu (0.5 mCi/0.1 ml) was introduced.Matrigel is expected to prevent internalization of trapped TiO₂ intocells. The cells were incubated at 37° C. for 48 h. Live/Dead® cellstains (Life Technologies Inc.) were used according to themanufacturer's instructions.

In Vivo Tumor Model.

Athymic nu/nu mice were purchased from Frederick Cancer Research andDevelopment Center. All studies were conducted in compliance withWashington University Animal Welfare Committee's requirements for thecare and use of laboratory animals in research. The HT1080 tumors weregenerated by subcutaneous injection of 4×10⁶ cells in 100 μl of DPBS inAthymic nude mice.

In Vivo Biodistribution Studies.

Athymic nude mice with a tumor volume of ˜300 mm³, were injected with 1mg/ml of TiO₂-Tf (n=5) or Tf alone (n=5) in 100 j.il of DPBSintravenously through tail vein, where Tf was labeled with Alexa 680 dye(Life Technologies Inc.). Fluorescence imaging was performed using anexcitation and emission wavelength of 685 nm and 720 nm, respectively,in a Pearl whole animal imager (Li-Cor Biosciences Inc.). The mice weresacrificed 24 h post-injection and the major organs were dissected andimaged. Mean fluorescence intensity was estimated by ROI analysis usingImageJ software. The intensity was normalized to equalize musclefluorescence levels and plotted for all the organs using Graph Pad Prismsoftware.

CRIT of Solid Tumors.

When tumor volume in mice reached 50 mm³, which is ˜7-9 days aftersubcutaneous implantation of cells, the mice (n=6 per group) wereinjected with 1 mg/ml TiO₂-Tf, Tc-Tf, TiO₂-Tf-Tc in 100 μl of DPBSintravenously and 0.87 mCi/0.1 ml of FDG also intravenously 48 h later.Control mice (n=6 per group) were administered with DPBS, theconstructs, or FDG alone. The mice were starved for 6 h beforeadministering FDG and kept in a dark room post injection, shielded bylead bricks. A second administration of FDG (0.87 mCi/0.1 ml) was given48 h after the first FDG injection. Similarly, two additional cohortswere administered with 0.14 mCi/0.1 ml and 0.43 mCi/0.1 ml FDG (n=4 pergroup), respectively, and monitored over 45 d. For intra-tumoraladministration, a cocktail of 2.5 μg/ml of TiO₂-PEG and 0.5 mCi/0.1 ml⁶⁴Cu in 50 μl of DPBS was injected directly into the tumor mass afterthe tumor volume reached 200 mm³ (˜12-14 days after subcutaneousimplantation of cells). Two diametrically opposite injection sites werechosen and 25 μl of the cocktail was delivered at each site. Four groups(n=4), TiO₂-PEG treated mice, ⁶⁴Cu treated mice, non-radioactive Cu (1μM CuCl₂) treated mice and untreated mice, served as controls. For both,systemic and intra-tumoral studies, the mice were monitored for 45 days.The growing tumors were measured with calipers every two days and tumorvolume (TV) calculated using the equation: TV=(length×width²)/2. The TVwas plotted versus time to analyze CRIT effect on the seed culture.Weight and any physical signs for distress were also monitored closely.Kaplan-Meir survival curves were plotted using Graph Pad Prism software.The mice with regressing tumors were monitored for an additional fourmonths to determine whether the cancer was in remission.

FDG-PET Imaging.

After anaesthetizing the mice with 1.5-2% Isoflurane and Oxygen, 0.19mCi (7.03 MBq)/0.1 ml FDG was administered i.v. A ten minute transitionscan was performed just before the ten minute emission at 1 h postinjection. The animals were placed on the microCT® in the same positionto obtain anatomical imaging that was co-registered to the microPET®image. The images were acquired using a MicroPET-Inveon MultiModalityscanner (Siemens Preclinical Solutions).

Histology.

The HT1080 tumor bearing mice in the control groups were sacrificed 15 dpost administration of constructs or FDG, while mice that underwent CRITwith TiO₂-Tf or Tc-Tf were sacrificed 30 d post administration, and thegroup that underwent CRIT with TiO₂-Tf-Tc at 45 d post administration.Likewise, for HT1080 tumor bearing mice, the mice were sacrificed 3 dafter intra-tumoral administration of TiO₂-⁶⁴Cu cocktail. The tumorswere harvested and snap-frozen in OCT media for routine staining withhematoxylin and eosin (H&E). Brightfield images of H&E stained 10 μmtumor sections were taken using the epifluorescent microscope at 4× and20× magnifications.

Statistical Analysis.

Unless noted otherwise, all values are means and error bars are standarddeviations. Statistical significance was measured by student T testusing Graphpad Prism software.

References for Examples 7-10

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Introduction to Examples 11-14

Phototherapeutic interventions such as photodynamic therapy (PDT) arecurrently used in clinics for cancer treatment. The exciting combinationof light and photosensitizer (PS) offers high degree of control that istypically used in different stages of cancer patient management as wellas other disease states. Despite the promise of PDT, the shallowpenetration of light in tissue confines its use to localized andsuperficial lesions. In addition, light dosimetry for effective PDTremains a challenge because of the difficulty in delivering externallight uniformly to the heterogeneous contours of diseased tissues. Asecond level of complexity arises from the efficacy of PS. Althoughthere has been significant progress in the development of newer andbetter PS drugs, other clinically and biologically relevant problemssuch as low sensitivity and selectivity, as well as sustained skinphotosensitivity, continue to diminish the effectiveness of PDT. Anothermajor limitation of current PS is the reliance on tissue oxygen togenerate cytotoxic singlet oxygen free radicals. This feature precludesthe effective application of PDT in the treatment of many solid tumors,which often have hypoxic regions.

We propose a two-prong approach that addresses the issue of shallowpenetration of light by employing Cerenkov radiation (CR) and the issueof tissue oxygen dependence and suboptimal activation of PS by usingpreviously unexplored light sensitive materials for effective CR-InducedTherapy (CRIT). CR from clinical grade radionuclides used in positronemission tomography (PET) emits predominantly a continuous spectrum ofultraviolet (UV) light. Particularly, the PET radionuclides Fluorine-18(¹⁸F), Copper-64 (⁶⁴Cu), and Zirconium-89 (⁸⁹Zr) emit CR suitable formolecular imaging applications by recording the weak visible lightradiance of CR. In addition, the clinical applicability of CR forquantifying disease signatures was recently demonstrated. To improve PSactivation, minimize reliance on molecular oxygen, overcome tissue depthdependency, and favor clinical translation, a new class of PSs capableof selective photoactivation using the low radiance from CR for CRIT isurgently needed.

Toward this goal, we have identified the light-sensitive materials,titanocene (Tc) and titanium dioxide (TiO₂) for CRIT. Tc is aphotoinitiator that can be activated by low intensity light to generatefree radicals, and titanium dioxide (TiO₂) is a regenerativephotocatalyst that produces free radicals capable of localizedcytotoxicity. There are several advantages to using Tc and TiO₂: 1. Thepredominantly hydroxyl radicals from TiO₂ and the photo-fragmentationproducts from Tc are generated in an oxygen independent fashion. 2. Theexcitation energy for Tc and TiO₂ are in the UV spectrum, where CRquantum efficiency is highest; the large surface area of TiO₂nanoparticles can efficiently harvest CR. These combined features favorCRIT. 3. Radical generation mechanisms for Tc and TiO₂ are different,which could result in different mechanisms of cell death. Thus, acombination of both materials could synergistically enhance treatmentoutcomes. 4. Transferrin (Tf), a tumor targeting protein, binds Tc withhigh affinity. Further, TiO₂ nanoparticles typically form aggregates inaqueous solutions, but addition of Tf produces monodispersed and stableTiO₂-Tf nanoparticles. Thus, treatment of TiO₂ with Tf spontaneouslyforms TiO₂-Tf, which readily binds Tc to generate TiO₂-Tf-Tc withoutloss of Tf tumor targeting affinity. This provides a simple method toprepare tumor-avid materials for both photoinitiator- andphotocatalyst-mediated CRIT. 5. Clinically, radiolabeled2′-deoxy-2′-(¹⁸F)fluoro-D-glucose (FDG) is widely used to image rapidlyproliferating cells, which can co-localize with TiO₂-Tf-Tc in tumorsoverexpressing Tf receptors. Our early results suggest that FDG caninduce effective CRIT with TiO₂-Tf-Tc. This is a first demonstration ofa strategy to overcome the low light intensity of CR. 6. The FDA hasapproved TiO₂ nanoparticles for use as colorant in food and as a UVprotective ingredient; Tc has been used in Phase II clinical trials as achemotherapeutic drug but was discontinued for lack of efficacy; and FDGis clinically used in PET. Therefore, the ensemble of products isclinically translatable using sublethal doses of both Tc and TiO₂.

This work should demonstrate the efficacy of CRIT in cancer therapy andshow that photoactivation of TiO₂-Tf-Tc by radionuclides such as¹⁸FDG-mediated CR will inhibit, reverse, or prevent tumor growth. Thiswork should answer the following questions: (1) Can a tumor-targetedphotoinitiator such as Tc inhibit tumor growth via CRIT? We expect thatphotofragmentation of Tc will produce free radicals that damage DNA,resulting in cell death. (2) Can a tumor-targeted photocatalyst such asTiO₂ inhibit tumor growth via CRIT? We expect cell death mediated bylight-induced and oxygen-independent hydroxyl radicals generated fromTiO₂ nanoparticles. (3) Can we achieve complementary CRIT by combingphotoinitiator- and photocatalyst-cell death mechanisms via TiO₂-Tf-Tcnanoparticles? We expect to observe a significant inhibition orregression of tumor growth with TiO₂-Tf-Tc than can be achieved withtumor targeted Tc or TiO₂ alone. We will determine the mechanism of celldeath in vitro and the rate of tumor growth or regression in vivo.

Example 11 Success and Struggles of Photodynamic Therapy (PDT)

Photodynamic therapy (PDT) is a viable treatment paradigm for cancer:PDT uses light of appropriate wavelengths to excite a photosensitizer(PS) in the tissue volume of interest. Upon light absorption, the PS canmediate PDT by Type I (direct transfer of radical ions from an activatedPS to biomolecules) or Type II (transfer of PS triplet state electronsto molecular oxygen, which generates reactive singlet oxygen species),or occasionally a combination of both mechanisms. The mechanisms of PDThave been reviewed extensively. The combination of non-lethal lightlevels and non-toxic PS to induce highly selective and localizedphototoxicity in target tissue remains an attractive feature of PDT, asreflected by its tremendous advancements over conventional cancertherapies. PDT has resulted in high cure rates for some tumors withoutcumulative toxicity. Examples of the diverse clinical uses of PDTinclude malignancies of the gastrointestinal tract, lungs, head & neck,and skin. To improve PDT efficacy, different approaches have beenemployed, including the conjugation of PS to carrier molecules fortumor-selective uptake, the development of new nanoparticles to amplifyradical generation, the use of nonlinear activation techniques toincrease treatment depth, and the design of activatable PS toselectively trigger photosensitivity in target tissue. Despite theselaudable accomplishments, the broad application of PDT in clinics hasbeen limited by several factors, some of which are summarized below.

Reliance on Molecular Oxygen for Effective PDT Precludes ApplicationUnder Hypoxic Conditions:

Regardless of the mechanism of action, both Type I and Type II PDTregimens currently used in the clinics rely on reactive oxygen productsfor therapeutic effect. This basic assumption implies that PDT will beless efficient under hypoxic conditions. Unfortunately, many solidtumors have significant hypoxic regions, some of which developresistance to PDT. Moreover, most PSs require high radiant exposure togenerate sufficient singlet oxygen species for PDT, which may harmsensitive healthy tissues, rapidly photobleach some PSs and possiblyproduce undesirable effects. Therefore, an oxygen-independentlight-induced therapy could address this fundamental problem.

PDT is Largely Confined to Localized and Shallow Lesions:

The limited penetrability of light in tissues and the challenges inoptimizing light dosimetry have prevented the full realization of theenormous potential of light-based imaging and therapeutic methods. BothUV and visible light (UV-vis) can only penetrate tissue from a fewmicrons to a few millimeters from the incident light, which has confinedtheir use to direct tissue ablation or the treatment of skin lesions. Toimprove the treatment depth, newer near infrared (NIR) absorbing PSshave been developed. Because NIR light can penetrate deeper in tissuethan UV-vis, these agents can improve the depth of treatment. Recentstudies have explored the use of multiphoton excitation of PS in the NIRwavelengths to activate PS in the visible light region. Unfortunately,this approach required PSs with high multiphoton absorption crosssections to harvest the multiphotons efficiently. Moreover, only smalltissue volumes can be treated per multiphoton event. Additionally, eventhese improvements can only interrogate tissue depths within 10 mm whenusing a high intensity light source. Further, the efficiency of lightdelivery decreases rapidly with tissue depth, requiring differentsettings to optimize depth-dependent dosimetry. This limitation can beovercome by the use of depth-independent light source to activate PS. Assuch, we have developed a molecular oxygen- and tissue depth-independentlight-induced cancer therapy, which will expand light treatment topathologies that are currently not amenable to current phototherapeuticmethods.

Titanium Based PSs can Eradicate Tumors in a MolecularOxygen-Independent Manner:

Titanium compounds are widely used in medicine, clean energy generation,and environment remediation because of their low toxicity, redoxactivity, and photoactive properties. Among them, Titanocene dichloride(Tc) and titanium dioxide (TiO₂) have shown promise as photoinitiatorsand photosensitizers, respectively. Tc is derived from the family ofmetallocenes and has been used in phase II clinical trials as achemotherapeutic drug. Although only mild to moderate side effects wereobserved at high doses, the clinical trials were discontinued due topoor treatment outcomes. Still, these studies established precedence forusing Tc in humans. Apart from its use in medical oncology, it is alsowidely used as a photoinitiator in the plastics industry. After exposureto UV light, Tc is able to generate free radicals in the presence orabsence of oxygen following photofragmentation. Because thephoto-initiation process occurs even with low radiance, and itsexcitation maximum is in the UV region (λ=250-325 nm),photofragmentation of sub-cytotoxic doses of Tc may induce DNA damageand subsequent cell death.

In addition to being used clinically in a variety of medical and foodformulations, previous studies have shown that TiO₂ nanoparticles areexcellent photocatalysts that can absorb UV light (λmax=275 nm) withhigh efficiency and generate free hydroxyl and superoxide radicalsthrough electron-hole pair production. Generation of hydroxyl radicalsthrough electron-hole transfer to chemisorbed H₂O is anoxygen-independent process, whereas superoxide radical generationrequires aerated aqueous media for electron transfer to molecularoxygen. Of these two products, the highly cytotoxic hydroxyl radicalsare the key species formed during the photocatalytic oxidation on thesurface of TiO₂ in aqueous solvents. These features have motivated theuse of TiO₂ as a PS to induce cell death in vitro. Moreover,biocompatible inorganic nanoparticles are attractive alternatives toconventional PS because of their large surface area, excellent payloadcapacity, and high reactivity. However, the shallow penetration of UVlight has confined most of the previous studies to in vitro models ofhuman diseases. The generation of cytotoxic free hydroxyl species at lowintensity UV light suggests that the low radiance of Cerenkov radiation(CR) can serve as a UV light source for depth-independentphotoactivation of the nanomaterials for phototherapy.

Cerenkov Radiation can Serve as Tissue Depth-Independent Light Sourcefor PDT:

Some clinically relevant radionuclides can produce a continuous spectrumof UV light via CR. CR occurs when charged particles such as positronsor electrons travel faster than the speed of light in a given medium,emitting predominantly UV light that tails off to the visible spectrum(250-600 nm). Positron emission tomography (PET) isotopes such asradiolabeled 2′-deoxy-2′-(¹⁸F)fluoro-D-glucose (FDG) are an ideal sourcefor CR because of their high positron (β+) emission decay and shorthalf-life. The β+ particles travel short distances (<1 mm) in tissues,during which CR is first emitted before they undergo annihilation.Recently, technological advances in low light detection techniques haveenabled the use of CR as a light source for molecular imaging. Werecently developed activatable CR probes for optical-nuclear imagingusing Copper-64 (⁶⁴Cu). Clinical application of CR imaging was recentlydemonstrated. Despite these advancements, CR remains a low intensitylight source, which limits the amount of material that can be activated,and thus requiring significant signal amplification and prolonged dataacquisition times to minimize background and dark noise. Our preliminarydata suggest that the low UV light threshold needed to excitephotoinitiators such as Tc and photocatalysts such as TiO₂ nanoparticlescould unleash a new paradigm in CR-Induced Therapy (CRIT).

There are several elements of innovation to this work: (1) The use of Tcand TiO₂ for CRIT for in vivo treatment of cancer in a depth-independentmanner is new. (2) The processing of crystalline TiO₂ to synthesizemonodispersed and tumor selective nanoparticles is new. (3) We havediscovered a new and efficient method to use transferrin (Tf) astumor-targeting agent, as well as Tc chaperone, TiO₂ chelator, a linker,and a dispersant to prevent nanoparticle aggregation. This approachsimplifies preparation of the PS for in vivo use. (4) We discovered thesynergistic effects of combining Tc and TiO₂ for efficient CRIT. This isthe first demonstration of the use of spontaneously generated CR at lowUV light intensity to inhibit tumor growth in depth-independent manner.(5) Because the components of the treatment methods are already used inhumans (Tc, TiO₂, Tf, and ¹⁸FDG), we envisage a clear path to humantranslation.

Although the Examples focus on specific photoinitiators and catalystsbecause of the limited time and resources to demonstrate feasibility,this work uncovers a new strategy to develop tailored molecularphotosensitive agents for treating cancer and other human diseases.Highly refractory tumors such as pancreatic cancer and gliomas, whichtypically require partial regression of tumor size before surgery, willbenefit highly from this technique. For example, intratumoraladministration of the photoactive agents and radionuclide cocktail willhelp achieve rapid tumor regression, as demonstrated in our preliminarystudies. In addition, hypoxic tumors that are resistant to radiationtherapy will now be sensitized for improved therapeutic outcomes becausethe CR from linear accelerators can activate the photoinitiators similarto PET radionuclides, opening new treatment techniques for thesepatients. Non-cancer diseases will also benefit from this method. Forexample, photoinitiators and catalysts can be targeted to bacteria toprevent infections during wound healing and minimize expensivereplacement of hip replacement transplants; purge latent HIV reservoirby HIV protease activation of photoinitiators; photo-stimulate neuronsto combat neurological disorders, etc. Taken together, this depthindependent PDT platform can be implemented to selectively inhibit oreradicate diverse diseases at cellular and tissue levels.

This work demonstrates the feasibility of using CR and highly sensitivephotosensitizers for depth-independent and highly selective CRIT.Although longer lived radionuclides with excellent CR such as ⁶⁴Cu, ⁹⁰Y,¹²⁴I, and ⁸⁹Zr, will be explored in future, this study will focus on FDGbecause it is trapped in cells with high metabolism, allowing uptake intumors without further modification. Similarly, many molecular designsfor delivering Tc and TiO₂ nanoparticles to tumors are available, but wewill focus on transferrin (Tf) for this study because it serves thetriple role of generating monodispersed TiO₂ nanoparticles, possesseshigh binding affinity for Tc, and delivers its cargo to tumors thatoverexpress Tf receptors. Together, the use of both Tf and FDG willaccelerate the proof of concept research, minimize product synthesis,and allow us to test parameters for detailed studies with more effectiveCR-radionuclides without loss of focus on the long-term translationalgoals of the project.

Example 12 Determination of CRIT Using Tc-Tf Bimolecular System

The goals of this Example are to (a) develop Tc-Tf adducts using Tf as atargeting ligand and binding site for Tc; (b) evaluate the tumorselectivity of Tc-Tf in tumor cells; (c) demonstrate CRIT effects intumor cells; (d) determine the mechanism of cell death; (e) determine invivo biodistribution and demonstrate tumor selectivity of Tc-Tf; and (f)demonstrate therapeutic response and long term survival in small animaltumor models. All animal and cell studies will be conducted with (i)HeLa cells, a human cervical cancer model, in which Tf receptorsinternalize rapidly after binding to Tf; and (ii) HT1080, a fibrosarcomamodel, in which Tf receptors internalize slowly after binding to Tf.These models will allow us to determine the use of the proposed platformin more than one tumor cell line. We describe the methods for HT1080cell line in the following sections. We used FDG activity of 32.5 MBq toensure that sufficient amount of the radionuclide was internalized bythe tumors. We will first administer different activities (2, 8 & 30MBq) of FDG and use PET imaging to determine the optimal therapeuticdose based on the lowest injected dose to maximize FDG activity in thetumor. We expect that this activity will be closer to the imaging dose.

Development of Tc-Tf Adducts:

Tc-Tf adducts will be prepared by adding an equimolar solution of Tc toApo-Tf. Due to the high affinity binding affinity of Tc to Tf, which issimilar to Tf-Fe(III), it is expected that the Tc-Tf adducts will bestable in a neutral buffer solution. The adducts will be purified usingmembrane filters and characterized by UV-vis spectrophotometry. Theabsorption spectrum is expected to reveal both Tf (λ=280 nm) and Tc(λ=322 nm) peaks. For binding assays, commercially available Alexa 680labeled Tf will be used to prepare the Tc-Tf adducts.

Determination of Tumor Selectivity of Tc-Tf:

Binding assays using Tc-Tf will be performed in live cells to determinethe binding capacity (B_(max)) and equilibrium dissociation constant(Kd) values, as described in the literature. We will conduct thesestudies in HT1080 cells, which overexpress Tf receptors. This cell linewill also be used for in vivo studies. Increasing concentrations of thefluorescent Tc-Tf constructs will be added to the confluent cellsfollowed by incubation at 37° C. and 4° C. for 2 h. Inhibition studieswith a 100-fold excess of unlabeled Tf will be used to determinenon-specific binding, which will be subtracted from total binding togive Tf-specific binding. We expect that the Kd of Tf and Tc-Tf will besimilar since Tc binding does not affect the binding to the Tf receptor.

Determination of Mechanism of Cell Death:

Tc is known to generate free radicals through photofragmentation onexposure to UV light and the nature of the radicals is wellcharacterized. Since Tc is known to intercalate DNA, we hypothesize thatupon UV illumination using CR, the free radicals will cause DNA strandbreakage, leading to apoptosis. We will perform agarose gelelectrophoresis to detect DNA fragments in lysed cells as well as aComet assay to determine DNA damage and fragmentation in cellulo. Wewill also assess the cytotoxicity profiles of Tc-Tf in vitro, with andwithout application of FDG, at therapeutically relevant escalating dosesin relation to different time intervals. Quantitative evaluation ofcellular cytotoxicity will be performed using independent assays thatassess various cellular parameters such as: 1. MTT, for measuringactivity of cellular enzymes and mitochondria using the Vibrant® MTTCell Proliferation Assay Kit through absorbance readings of samplecells/control cells. 2. Propidium iodide staining of cells, specific todouble stranded DNA and indicative of cell membrane integrity, will becarried out using the Coulter® DNA Prep™ Reagents Kit and analyzed usingflow cytometry. In addition, we will also determine the mitotic index ofthe respective cells, to count the number of mitotic cells as anindicator of mitotic arrest and impending cell death. 3. Detection ofmono and oligonucleosomes in the cytoplasm, indicative of endogenousendonuclease activation in apoptotic cells, will be carried out usingCell Death ELISA^(PLUS) kit through absorbance readings of samplecells/control cells. Dose vs. response will be plotted for these assaysand data will be statistically analyzed using Graph Pad Prism software.LD₅₀ (dose that kills 50% of the cells) values for the constructs willbe determined from a plot of percentage cell death vs. Tc-Tfconcentration using fixed FDG activity. We will observe and monitorcellular parameters for apoptosis over a period of 5 days. Based on ourpreliminary studies, we expect LD₅₀ to be achieved at Tc concentrationat least 4-fold lower with CR than without FDG.

Determination of CRIT in Tumor-Bearing Mice:

We will use the Alexa 680 fluorescent Tf-Tc to determine the optimaltime point for CRIT by fluorescence imaging at different time points(0.5, 2, 4, 8, 24, 48 and 96 h) post-injection using the LICOR Pearlimaging system with 685/720 nm excitation/emission, as we reportedpreviously. HT1080 tumors will be implanted subcutaneously and treatmentwill be initiated in three phases (1) five days post injection of tumorcells when the tumors are barely palpable, to determine the feasibilityof eradicating tumors in early stages of growth; (2) when tumor massreaches 5 mm, to assess the feasibility of regressing tumor growth; and(3) when tumor mass reaches 10 mm, to assess the feasibility ofinhibiting tumor growth and defining tumor boundaries for accuratesurgical tumor resection, especially in highly sensitive organs such asthe brain and to minimize margin positivity, thereby minimizing patientrecall rates. Based on the time point of highest tumor:liver contrast(the expected major excretion organ of Tf-Tc) in vivo, the mice will beeuthanized, and major organs exhibiting Alexa 680 fluorescence will beharvested and imaged ex vivo to confirm in vivo data. Organs will thenbe separately homogenized and the product will be extracted with 40%DMSO in PBS for quantitative analysis of the Tf-Tc distribution. We willreport the distribution as percent injected dose/g organ.

The HT1080 tumor bearing mice will be randomly assigned to two cohorts,for each phase specified above, of 5 mice per group: (i) untreatedcontrol; (ii) FDG treated control; (iii) Tc-Tf (1 mg/kg) treatedcontrol; and (iv) Tc-Tf and FDG treated group. Using the optimal timepoint for tumor-to-liver uptake of Tc-Tf for the different tumor sizes,we will administer Tc-Tf as a single dose intravenously. Two FDG doseswill be administered on alternate days to account for the shorthalf-life of the ¹⁸F isotope. The body weights and tumor volumes will bemeasured thrice a week in each group. Caliper measurements will be usedto calculate tumor volumes (IV) using the equation: V=7/6(length×width²). The percentage of tumor growth inhibition will becalculated as 100×(mean TV of treated group)/(mean TV of untreatedcontrol group). Statistically significant differences in tumor volumesbetween control and drug-treated mice will be determined by theMantel-Cox test.

The first cohort of each phase will be euthanized at day 7, afterinjections, to evaluate the acute therapeutic parameters such as cellproliferation (e.g. Ki67 immunohistochemistry), decreased microvasculardensity and apoptosis (e.g. TUNEL IHC) in tumor tissue, throughhistopathology. Measurement of acute inflammatory signs such as fluidaccumulation and identification of neutrophils in the affected site willbe carried out using histopathologic assessment. The second cohorts willbe monitored for effect of therapy on long-term tumor growth.Kaplan-Meier survival analysis will be carried out to measure thefraction of mice living for a certain amount of time after treatment.Mice will be euthanized when tumor size reaches 1.5 cm maximum diameteror lose >10% body weight. Associated complications of high dose rates onall major organs and nearby tissues will be evaluated. Histochemicalanalysis of the tumors, including H&E staining and Ki67immunohistochemistry, will be performed by counting the number of Ki67positive and negative cells in ten randomly chosen areas of the tissuesections from each treatment and control group. We expect to achievefaster tumor regression for the constructs with the lower administereddose, without inducing any acute inflammatory changes to vital organs.

Histology:

Histologic validation of tumor death will be performed by histologicalsection analysis.

In the event of unstable interaction between Tc and Tf, we willcovalently link Tc to antibodies such as anti-epidermal growth factorreceptor antibody and perform stability and binding studies. The use ofAlexa 680 labeled Tf as a surrogate for the distribution of Tc-Tf isbased on the strong binding of Tc to Tf until it dissociates within theacidic intracellular lysosomes before translocating to the nucleus. Astudy showed that the biodistribution of the radiolabeled Tc-Tf analogue(⁴⁵Ti-Tf) was consistent with Tf distribution. If treatment responsedoes not correlate with the determined fluorescence biodistributionprofile, we will prepare a stable scandocene analogue, which can bereadily converted to radiolabeled Tc. The initial doses for Tc-Tf andFDG are based on published sub-lethal doses of Tc¹⁹ and an exploratorydose of FDG, respectively. In the event of unappreciable tumor volumereduction, an increase in the administered doses will be considered.

Example 13 Determination of CRIT Using TiO₂-Tf Bimolecular System

Development of TiO₂-Tf Adducts:

TiO₂ typically exists in two tetragonal forms, anatase and rutile, whichdiffer in their crystal lattice structure. We will employ the anataseform for CRIT studies because of its smaller size and higherphotoactivity arising from the extensive surface hydroxyl groups inwater. Larger or smaller-sized monodispersed nanoparticles can beobtained by starting with different sized nanocrystals. We will startwith commercially available ˜25 nm crystalline TiO₂ and use our newlydiscovered Tf formulation to create monodispersed nanoparticles of ˜18nm from the crystalline TiO₂ aggregates. In this method, a suspension ofTiO₂ and Tf will be sonicated using a probe sonicator and immediatelyfiltered through membranes. By using membrane filters of different porecutoff points such as 0.1, 0.2 and 0.4 μm, we can narrow the sizedistribution. Membrane dialysis will be used to remove unbound Tf fromthe TiO₂-Tf adducts. UV-vis, TEM and DLS particle analysis will beperformed on each batch. Determination of tumor selectivity of TiO₂-Tfwill be as described in Example 12.

Determination of mechanism of cell death will be as described in Example12. Assays will be performed to evaluate whether the mechanism of celldeath is through apoptosis or necrosis. TiO₂ generates cytotoxichydroxyl and superoxide radicals when irradiated with UV light. We willquantitatively estimate the amount of hydroxyl and superoxide radicalsusing fluorescent dyes such as hydroxyphenyl fluorescein and Mitosox,respectively. Blocking studies with L-Tryptophan, for hydroxyl radicals,and superoxide dismutase, for superoxide radicals, will be performed.

Determination of CRIT in Tumor-Bearing Mice:

Using Alexa 680 labeled Tf, biodistribution and tumor specificitystudies of TiO₂-Tf adduct will be carried out as described in Example12, as well as efficacy studies of using TiO₂-Tf as a photocatalyst forCRIT. Histology will be as described in Example 12.

The stability of TiO₂-Tf in solution and the potential forre-aggregation over time is a concern. TEM and DLS analyses performed12-14 h after synthesis suggest minimal re-aggregation and no noticeablechange in the polydispersity index. However, we plan on performingstability studies, using TEM and DLS, both in buffer solution and serumover extended periods—up to a month post-formulation. If long-termstability is a problem, we will prepare new batches after the useableshelf-life.

Example 14 Determination of CRIT Using Tc-TiO₂-Tf Trimolecular System

Development of TiO₂-Tf-Tc:

To prepare TiO₂-Tf-Tc, we will use the Tc-Tf prepared in Example 12 toprepare the monodispersed TiO₂-Tf-Tc following the procedure describedin Example 13.

For determination of tumor selectivity of TiO₂-Tf-Tc, we will usesimilar methods as described in Example 12.

For determination of mechanism of cell death, we will use the samemethods described in Example 12 for this study. We expect to observe acombination of photoinitiator- and photocatalyst-induced cell deathmechanisms. We will determine if the effect is additive or synergisticby comparing the LD₅₀ of CRIT for TiO₂-Tf-Tc relative to equalconcentrations of TiO₂-Tf and Tc-Tf under the similar conditions.

Determination of CRIT in Tumor-Bearing Mice:

We will perform CRIT with TiO₂-Tf-Tc using the procedure described inExample 12. We will evaluate if the treatment response is an additive(complementary) or amplified (synergistic) effect based on theinhibition of tumor growth rate relative to equal concentrations ofTiO₂-Tf and Tc-Tf under similar conditions.

Histology:

We will use a similar method as described in Example 12.

In the event there is unstable association of Tc to TiO₂-Tf, we willcovalently conjugate Tc directly to the surface of TiO₂ using suitableintracellular cleavable linkers such as disulfide bonds to allow releaseof Tc for subsequent translocation to the nucleus under the highlyreducing intracellular environment of tumor cells.

Example 15 Systemic CR-PDT

After achieving successful tumor regression in a fibrosarcoma modelthrough intratumoral administration of TiO₂ and ⁶⁴Cu, we developed aclinically relevant strategy to achieve targeted CR-PDT through systemicadministration of the PS and radionuclide. Synthesis of monodisperse,ultrafine spherical TiO₂ with narrow size distribution suitable forsystemic administration using inorganic titanium salt remains achallenge. Here, we demonstrate for the first time a “green” strategy toachieve monodisperse TiO₂. The strategy includes development of hybridTiO₂ conjugates with a targeting moiety, transferrin (Tf)—a ubiquitousiron transporter found in serum. Many tumors including HT1080fibrosarcoma overexpress transferrin receptors due to high demand foriron by rapidly proliferating cells. TiO₂-Tf conjugates were furtherappended by a biocompatible photoinitiator, titanocene (Tc), to enhancethe generation of free radicals and improve cytolytic activity of theconjugates. Tc is a visible light photoinitiator and TiO₂ a UVphotosensitizer, therefore, consolidating their use in conjunction withCR that has a broad emission spectrum spanning UV and visiblewavelengths, will only enhance the overall design. Thephotoinitiator-photosensitizer two component system interact throughboth energy transfer and electron transfer mechanisms, complementingeach other and consequently exhibiting faster and higher radicalgeneration. Like iron, Tc has high binding affinity to apo-Tf, andtherefore readily forms a stable complex with TiO₂-Tf. TiO₂-Tf-Tccomplexes as next-generation PDT agents, can therefore offer highlyefficient radical species generation through synergistic activity of thetwo photoactive components and at the same time also offer targetingfunctionality towards Tf receptor expressing tumors.

In vivo biodistribution studies carried out in 4T1 and HT1080 tumorbearing mice using Alexa 680 labeled Tf-TiO₂ conjugates, show highestuptake in the tumors (FIG. 21). The tumor:muscle ratio's registered forTiO₂-Tf and Tf alone were 9 and 5.5, respectively. Biodistributionstudies carried out over 96 h suggest gradual clearance of theconjugates through the hepatobiliary and renal system. Since the highesttumor:background contrast was obtained at 24 h post injection,accordingly this was chosen as “drug-light interval”. As CR source, aradionuclide with high tumor selectivity and uptake is desired toachieve optimum activation of the TiO₂ constructs.F-18-fluorodeoxyglucose (FDG) is ideal because of its affinity towardsrapidly proliferating cells, which affords it high tumor selectivity, aswell as its high energy (633 keV) positron (97%) emission whichgenerates CR with high fluence rate. Moreover, FDG has a reasonablyshort half-life (109 min) that is conducive for avoiding systemictoxicity in the short term, which is a result of residual PDT occurringin non-targeted tissues. It's current clinical utility for highresolution PET imaging and monitoring therapeutic response is an addedadvantage.

After intravenous administration of the TiO₂ constructs and FDG intomice bearing HT1080 tumors, the animals were monitored over 15 days(FIG. 22). It was observed that the tumor progression rate for the micetreated with TiO₂-Tf, TiO₂-Tf-Tc and Tc along with FDG was considerablyslower in comparison to mice that were left untreated as well as treatedwith only the constructs or FDG, as controls (FIG. 23). The tumor volumeregistered for mice treated with TiO₂-Tf and Tc with FDG was four-foldsmaller compared to the controls. Whereas, mice treated with TiO₂-Tf-Tcand FDG showed a superior response to the treatment with a tumor volumeof eight-fold smaller compared to controls. This suggests thesynergistic activity of TiO₂ and Tc in achieving better treatmentresponse is a result of amplified generation of free radicals. Theattenuated growth rate of tumors after systemic administration of theconstructs is significant because the dose of the constructs wasmaintained at sublethal levels, known to not cause any dark toxicity;and activity of FDG injected was that of clinically acceptable levelsroutinely used in small animal imaging studies.

Methods for Example 15 Synthesis of TiO₂-Tf and TiO₂-Tf-Tc Conjugates

Human transferrin from Sigma Aldrich (St. Louis, Mo.) was dissolved inPBS at a concentration of 1 mg/ml to which 500 mg/ml of TiO₂ was added.After brief sonication, using a probe sonicator, the solution wasfiltered using a 0.4 mm syringe filter to obtain TiO₂-Tf conjugates. Theconjugates were further purified using 100 kDa MWCO membrane filtersfrom Millipore, to remove unbound Tf. The conjugates were analyzed byUV-vis spectrophotometer before and after filtration to estimate yieldof the final conjugates. A working stock solution of 5 mg/ml Titanocenedichloride from Sigma Aldrich was prepared in DMSO. To a solution ofTiO₂-Tf, 100 mg/ml of Tc was added and incubated at RT for 1 h. UnboundTc was removed from TiO₂-Tf-Tc constructs using 3 k MWCO membranefilters.

Biodistribution Studies:

To track TiO₂-Tf conjugates, Alexa 680 labeled Tf was used. The studieswere commenced when tumor volume reached 500 mm³. 0.5 mg/kg of TiO₂-Tfwas injected intravenously in mice with 4T1 (n=3) and HT1080 (n=3)tumors and monitored for 96 h post injection using Li-cor Pearl animalimaging system with an Ex/Em of 680/715 nm. The animals were euthanizedsubsequently and major organs dissected and imaged in the same channel.Image J was used to quantitate fluorescence intensity by drawing regionsof interest encompassing entire organs.

Systemic CR-PDT:

After the HT1080 tumors grew to ˜20 mm³, 1 mg/kg of TiO₂-Tf wasadministered intravenously. After 24 h, 1 mCi of FDG was administeredintravenously. Tumor volume was registered every alternate day for 15days.

Example 16 Physical Characterization of TiO₂-Tf

A strong signature of carbon in the Energy-dispersive X-ray spectroscopy(EDX) spectrum of TiO₂-Tf compared to TiO₂ alone (FIG. 24A) confirmedthe presence of Tf on the surface of TiO₂ (FIG. 24B). Electrondiffraction analysis confirmed that the crystal lattice structure ofanatase TiO₂ remained unchanged as a result of processing with Tf (FIG.24C,D). Phase transformation between anatase and rutile forms of TiO₂typically occur at temperatures exceeding 700° C. Due to the relativelymild nature of the processing used to generate TiO₂-Tf adducts, thephotocatalytic properties of anatase TiO₂ employed in this study wasthus maintained. The ring measurements are as follows:

TiO₂—

5.7 1/nm/2=0.351 nm=3.51 A

8.4 1/nm/2=0.238 nm=2.38 A

10.51/nm/2=0.189 nm=1.89 A

11.81/nm/2=0.169 nm=1.69 A

TiO₂-Tf—

5.7 1/nm/2=0.351 nm=3.51 A

8.4 1/nm/2=0.238 nm=2.38 A

10.5 1/nm/2=0.190 nm=1.90 A

11.8 1/nm/2=0.169 nm=1.69 A

Example 17 Serum Stability of TiO₂-Tf

To determine the serum stability of the TiO₂-Tf interaction, weincubated the nanoparticles in fetal bovine serum for 24 h. We foundthat the amount of bound AlexaTf to TiO₂ surface did not significantlychange with time (FIG. 25A). Further analysis of data showed that serumcomponents such as albumin, using Alexa 680 labelled BSA, did not formappreciable protein corona on the TiO₂-Tf surface relative to pristineTiO₂ (FIG. 25B), confirming that Tf does not readily exchange with serumproteins.

Example 18 In Vitro and In Vivo Blocking Study Using TiO₂-Tf Labelledwith Alexa 680

Competitive inhibition of TiO₂-Tf internalization in tumour cells usingsaturating amounts of holo-Tf (iron-chelated Tf; FIG. 26) demonstratesspecific Tf-mediated endocytosis. However, attempts to reproduce thisresult in vivo only showed noticeable but statistically insignificantreduction in tumour uptake of the NPS (FIG. 27). We attribute thisfinding to several factors, including the high turnover rate of Tfreceptor after endocytosis, difficulty in saturating Tf receptor in vivowith saturating dose of Tf, and the high avidity of the TiO₂-Tf adduct.

Example 19 In Vitro Assay to Determine Peroxyl Radical Generation fromPhotofragmentation of Titanocene

Earlier reports have suggested the formation of peroxyl radicals by thephotofragmentation products. To determine if the peroxyl radicals causeperoxidation of cellular lipids, a BODIPY® 581/591 C11 reagent wasemployed. While Tc and FDG induced significant peroxidation anddegradation of cellular lipids through the free radicals, addition ofperoxyl radical scavengers, such as Trolox, adequately inhibited thisprocess (FIG. 28). This suggests a strong correlation between lipidperoxidation and photofragmentation of Tc through CR from FDG,implicating peroxyl radicals as the affector. Treatment with2,2′-Azobis(2-methylpropionamidine) dihydrochloride (AAPH) was used asthe positive control.

Example 20 In Vitro Assessment of Mitochondrial Membrane Potential as aResult of CRIT

To further understand the role of free radicals and the mechanism ofcell death, we evaluated whether CRIT induces alterations inmitochondrial membrane potential. There was a significant decrease inmitochondrial membrane potential in cells treated with both TiO₂ and Tccoincubated with FDG, indicating damaged and leaky membranes (FIG. 29).Typically, damage to mitochondrial membranes initiates the intrinsicsignalling pathway for apoptosis, characterized by loss of membranepotential and a cascade of events involving caspases leading to nuclearfragmentation and cell death.

Example 21 TEM Analysis of Tumor Uptake of TiO₂-Tf-Tc

To determine internalization of the non-fluorescent TiO₂-Tf-Tcconstructs in tumour cells after systemic administration, we employedTEM for the ex vivo analysis of tumour sections The TEM images of tumoursections clearly show TiO₂ nanoparticles as dark spots in the cells,demonstrating the tumour uptake of the TiO₂-Tf-Tc NPS in majority of thecells and the retention of monodispersity in vivo (FIG. 30A). Inaddition to Tf-mediated endocytosis, the monodisperse, small size, andfavourable surface properties of the Tf adducts probably facilitated thetumour uptake via enhanced permeability and retention (EPR) effect. Thefractional contribution of EPR and avidity effects could be gleaned fromthe differences in the tumour-to-muscle ratio of 5.3 for Tf alone, whichis much lower than that of TiO₂-Tf. In contrast to Tf-facilitatedendocytosis, the tumour cells did not appear to internalize TiO₂-PEGaggregates in vivo (FIG. 30B), suggesting that the observed peritumouraluptake of these particles was primarily mediated by EPR effect.Therefore, a combination of both extracellular (EPR) and intracellular(Tf) processes accounts for the higher accumulation of TiO₂-Tf-Tc in thetumour environment than TiO₂-PEG nanoparticles.

Example 22 Analysis of CRIT In Vivo

Comparison of untreated (FIG. 31A) and treated (FIG. 31B) tumoursections using TEM shows predominantly apoptotic cells in the latter.The localization of TiO₂-Tf based constructs in the apoptotic (FIG. 31C)and necrotic (FIG. 31D) cells also confirms the selectivity of themethod.

Assessment of body weight and remission after achieving CRIT inducedregression of tumors revealed that CRIT-treated animal steadily gainedweight whereas untreated animals quickly declined (FIG. 32).

Next, in vivo CRIT was evaluated in an A549 lung tumor model. Treatedmice received TiO₂-Tf-Tc and FDG. There was a significant reduction intumor burden in the treatment mice relative to the untreated mice (FIG.33).

Additionally, in vivo CRIT was evaluated in the U266 multiple myelomatumor model. U266 subcutaneous xenograft tumors were grown in NSG miceand treatment was initiated once the tumors became palpable. An i.v.dose 1 mg/kg of nanoparticle construct was administered, followed by ani.v. dose of 31 MBq/0.1 mL of ¹⁸FDG after 24 h. The mice received repeatinjections on day 6 and 12. We observed that the tumor growth rate forthe mice undergoing CRIT was considerably lower than in untreated mice.Median survival increased from 13±2 d, for the untreated and controlgroups, to 21.5±3 d for mice treated with construct and ¹⁸FDG (FIG.34A,B). SPEP analysis revealed considerably lower γ-globulins in miceundergoing CRIT (FIG. 34C), compared to untreated mice, suggesting adecrease in tumor burden. CRIT-treated tumors had a significantly lowerGFP fluorescence (FIG. 34E) than untreated tumors (FIG. 34D), suggestingthe presence of predominantly dead cells in the tumor matrix. Althoughnone of the experimental protocols were optimized at the time, theresults clearly demonstrate the efficacy of CRIT in inhibiting MMproliferation and extending survival. We now plan to optimize thetreatment protocol and nanoparticle size for clinical translation.

Methods for Examples 16-22 Synthesis of TiO₂-PEG TiO₂-Tf, Tc-Tf andTiO₂-Tf-Tc

Anatase TiO₂ (1 mg; Sigma Aldrich Co., St. Louis, USA) was suspended indeionized water (1 ml) to prepare working stock solution. PEG 400 (100μl) was added to the TiO₂ solution and sonicated using a probe sonicatorfor 10 min at room temperature (RT). The mixture was then dialyzedovernight against Dulbecco's Phosphate Buffered Saline (DPBS) using a3000 Da molecular weight cutoff Slide-A-Lyzer MINI Dialysis Devices(Thermo Fisher Scientific Inc., Waltham, USA) to remove excess PEG.Working stock solutions of Tf were prepared by dissolving 5 mg of humanapo-Tf (Sigma Aldrich Co.) in 1 ml DPBS, pH 7.4. To prepare TiO₂-Tf, a1:1 (v/v) solution of TiO₂ and Tf was mixed and probe sonicated incontinuous mode for ˜2 min. It is important to ensure the temperature ofthe solution does not exceed 55° C. to prevent denaturation of Tf (60°C.). The solution was then immediately passed through a 0.45 μm syringefilter to isolate monodisperse nanoparticles. To prepare Tc-Tf,five-fold molar excess of Tc (Sigma Aldrich Co.) was added to humanapo-Tf and incubated in a shaker for 2 h at room temperature (RT). Aworking stock of Tc was initially prepared in DMSO due to low solubilityof Tc in water and aqueous buffers. The mixture was then dialyzedovernight against DPBS using a 3000 Da molecular weight cutoffSlide-A-Lyzer MINI Dialysis Devices to remove excess Tc. TiO₂-Tf-Tc wassimilarly prepared by incubating Tc with TiO₂-Tf conjugates andthereafter dialyzed to remove excess Tc.

Physicochemical Characterization:

TEM images of TiO₂ based constructs were acquired using a FEI TecnaiSpirit Transmission Electron Microscope (FEI, Hillsboro, USA) operatingat an acceleration voltage of 200 kV. EDX and electron diffractionanalysis was performed using a JEOL 2000FX TEM (JEOL USA Inc., Peabody,USA) and Philips EM420 TEM@120Kv (TEM Analysis Service Lab, Azle, USA).TEM grids coated with a layer of formvar were used throughout thesestudies. Dynamic light scattering measurements were acquired with aMalvern Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, UK)instrument equipped with a 633 nm laser. Three measurements wereconducted for each sample with at least 10 runs and each run lasting 10s. All sizes reported were based on intensity average. Absorptionspectra of TiO₂ and Tc were recorded on a Beckman Coulter DU 640UV-visible spectrophotometer (Beckman Coulter Inc., Brea, USA) andanalysed using Graphpad Prism statistical software. Fluorescence spectraof TiO₂ were recorded on a Fluorolog-3 spectrofluorometer (Jobin YvonHoriba, Kyoto, Japan). The sample was placed in a quartz cuvette andmeasurements recorded in triplicates.

Cell Culture:

All cell lines were purchased from American Type Culture Collection(ATCC, Manassas, USA) that underwent STR profiling and tested formycoplasma contamination. HT1080 fibrosarcoma cell line was culturedunder recommended standard conditions. HT1080 cells were cultured inDulbecco's Modified Eagle's Medium containing 10% foetal bovine serum(FBS), L-glutamine (2 mM), penicillin (100 units/ml), and streptomycin(100 μg/ml), incubated at 37° C. in a humidified atmosphere of 5% CO₂and 95% air. For cytotoxicity studies, a concentration of 2.5 pg/ml ofthe TiO₂-Tf, Tc-Tf, and TiO₂-Tf-Tc constructs as well as 0.2, 0.4 and0.85 mCi/0.1 ml of FDG; and 0.5 mCi/0.1 ml ⁶⁴Cu were used. Randomizedblock design was used for all in vitro experiments, which were run intriplicates, by dividing them into three blocks for all the treatmentgroups.

In Vitro Cell Viability Assays:

MTS(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)assay, a calorimetric assay for assessing viability of cell culture, wasperformed using CellTiter 96® AQ_(ueous) Non-Radioactive CellProliferation Assay kit (Promega Co., Madison, USA) according to themanufacturer's instructions. The cells were incubated with theconstructs and FDG for 48 h before analysis.

Alkaline comet assays (Cell Biolabs Inc., San Diego, USA) were performedusing the manufacturer's protocol. Briefly, treated and untreatedcontrol cells were removed from flask by scraping with a rubberpoliceman. The cell suspension was centrifuged and washed with ice-coldDPBS two times and re-suspended at 1×10⁵ cells/ml in ice-cold DPBS.Cells were embedded in low melt Comet agarose and plated on providedmicroscope slides. The cells were then lysed with lysis buffer andtreated with alkaline solution. The slides were electrophoresed inalkaline solution at 1 V/cm with a setting of 300 mAmp for 30 minutes.The slides were stained with Vista Green DNA dye after washing anddrying. Fluorescence images were acquired using an Olympus BX51epifluorescence microscope equipped with a charge coupled device camera.% Tail DNA was estimated using OpenComet (v1.3) plugin for Image Jsoftware.

TEM Analysis of Cells and Tissue with TiO₂-Tf and Tc-Tf:

For ultrastructural analysis, cells and tissue samples were fixed in 2%paraformaldehyde/2.5% glutaraldehyde (Polysciences Inc., Warrington,USA) in cacodylate buffer (100 mM, pH 7.2) for 1 h at room temperature.Samples were washed in cacodylate buffer and postfixed in 1% osmiumtetroxide (Polysciences Inc.) for 1 h. Samples were then rinsedextensively in distilled water before en bloc staining with 1% aqueousuranyl acetate (Ted Pella Inc., Redding, USA) for 1 h. Following severalrinses in water, samples were dehydrated in a graded series of ethanoland embedded in Eponate 12 resin (Ted Pella Inc.). Sections of 95 nmwere cut with a Leica Ultracut UCT ultramicrotome (Leica MicrosystemsInc., Buffalo Grove, USA), stained with uranyl acetate and lead citrate,and viewed on a JEOL 1200 EX transmission electron microscope (JEOL USAInc.) equipped with an AMT 8 megapixel digital camera (AdvancedMicroscopy Techniques, Woburn, USA).

In Cellulo Receptor Binding:

We used Tf labelled with Alexa 680 (AlexaTf; Life Technologies,Carlsbad, USA) to prepare fluorescent TiO₂-AlexaTf construct, and theproducts were processed as described above. HT1080 cells were grown in 8well chamber slides and incubated with TiO₂-AlexaTf, final concentrationof 0.25 mg/ml, and incubated for 1 h at 37° C. For Tf blocking, 25 mg/ml(100×) of holo-Tf (Sigma Aldrich Co.) was added and incubated for 1 hbefore adding TiO₂-AlexaTf. The wells were washed 3× before imaging.Fluorescence images were acquired using an Olympus FV1000 confocalmicroscope. Fluorescence/brightfield cell images were taken with a 60×objective using a He:Ne 633 nm excitation laser and emission range ofdichroic mirror set to 655-755 nm. Fluorescence and brightfield imageoverlay with false colour was performed using Fluoview FV10-ASW softwarefrom Olympus (Center Valley, USA). One hundred cells per well werecounted to quantify fluorescence intensity.

In Cellulo Hydroxyl and Superoxide Radical Assay:

Hydroxyphenyl fluorescein (HPF) with an excitation and emissionwavelength of 490 nm and 515 nm, respectively (Life Technologies Inc.)was used according to the manufacturer's instructions. Briefly, the 5 mMstock was diluted 1,000× to prepare 5 μM working stock solution in DPBS.The TiO₂-Tf, Tc-Tf and TiO₂-Tf-Tc and FDG treated HT1080 cells grown in8 well culture slides were immersed in the HPF working stock 4 h posttreatment. The cells were incubated for 1 h before the dye solution wasremoved and replaced with fresh DPBS. The cells were imaged byconfocalmicroscopy using the 488 nm Argon ion laser with emission set to 500-600nm. Similarly, Mitosox Red (Life Technologies Inc.) with an excitationand emission wavelengths of 510 nm and 580 nm, respectively, was used todetect superoxide radicals according to the manufacturer's instructions.

Lipid Peroxidation and Mitochondrial Membrane Potential Assay:

BODIPY® 581/591 C11 reagent (Life Technologies Inc.) was used toquantitatively determine the degree of lipid peroxidation. It is aratiometric fluorescence technique that relies on oxidation of lipids toshift fluorescence emission peak from 590 nm to 510 nm. HT1080 cells(˜10,000) were grown in 96 well plate and incubated with Tc-Tf (2.5μg/ml) and FDG (0.85 mCi/0.1 ml) for 6 h. Peroxyl radical scavenging wasperformed by coincubating Tc-Tf+FDG with Trolox (1 mM), a water solubleanalogue of Vitamin E and a powerful antioxidant. As positive control,2,2′-azobis-2-methyl-propanimidamide, dihydrochloride (AAPH; 1000) wasused. All the compounds were incubated for 6 h at 37° C. Finally, theBODIPY-based dye (100) was added and incubated for 30 min at 37° C.After washing the cells with PBS three times, the plate was analysedusing a Synergy HT multimode plate reader (BioTek Instruments Inc.,Winooski, USA) with excitation/emission of 581/591 nm for the reduceddye and at 488/510 nm for the oxidized dye. The ratio of thefluorescence intensities at 590/510 nm was plotted to derive the ratioof fluorescence change.

For measuring mitochondrial membrane potential, Mitotracker Green (LifeTechnologies Inc.) was used according to manufacturer's instructions.Staurosporine (2 μM; Sigma Aldrich Co.) was used as positive control.Fluorescence readout was performed using a plate reader usingexcitation/emission wavelengths of 490/516 nm.

Matrigel Based Cell Studies:

Matrigel™ (BD Biosciences, San Jose, USA) was thawed at 4° C. Forentrapment of NPS, we first mixed an equal volume of TiO₂ NPS solutions(3 mg/ml) with Matrigel, which was then plated on 8 well chamber cultureslides. HT1080 cells were then introduced on the slides and allowed togrow in Matrigel. Since Matrigel solidifies at 37° C., TiO₂ NPS remainsuspended and immobilized in the gel and are not internalized by thesurrounding cells. For the internalization model, Matrigel was omittedand cells were incubated with TiO₂ NPS to facilitate internalization.The cells were then washed to remove non-internalized TiO₂ NPS andreseeded on chamber slides. ⁶⁴Cu (0.5 mCi/0.1 ml) was then added to therespective chambers with (extracellular TiO₂) and without (intracellularTiO₂) matrigel and incubated for 48 h at 37° C. before performing theLive/Dead assay (Life Technologies Inc.) according to the manufacturer'sinstructions.

Chelation of ⁶⁴Cu to DOTA: For experiments with ⁶⁴Cu, we prepared astock solution (1 mg/ml) of DOTA(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; MacrocyclicsInc., Dallas, USA) in ammonium acetate buffer (50 mM) equilibrated to pH5.5. Aliquots of DOTA stock solution (50 μl) was added to ammoniumacetate buffer (450 μl), followed by ⁶⁴Cu (5 mCi; 185 MBq) in 0.1 Mhydrochloric acid (5 μl). The reaction mixture was incubated at 45° C.for 1 h in a shaker. Non-chelated ⁶⁴Cu was removed from the chelatedDOTA-⁶⁴Cu using a Waters HPLC purification system. The flow rate was setto 1 ml/min. The solvents were A: 0.1% trifluoracetic acid (TFA) inwater, and B: 0.1% TFA in acetonitrile. After 5 min hold at 5% B, thegradient was programmed linearly to 100% B at 40 min. The sample elutedat 6 min post injection, corresponding to the peak in the radiometer andUV detector. The sample was then dried in a rotary shaker to remove TFAand acetonitrilebefore re-suspending the residue in DPBS.

In Vivo Tumour Model:

Athymic nu/nu mice (8 week, female) were purchased from Frederick CancerResearch and Development Center (Frederick, USA). All studies wereconducted in compliance with Washington University Animal WelfareCommittee's requirements for the care and use of laboratory animals inresearch. The HT1080 xenografts were generated by subcutaneous injectionof 4×10⁶ cells in DPBS (100 μl) in both flanks of Athymic nude mice.Likewise, a bilateral subcutaneous tumour model of A549 was developedusing 5×10⁶ cells in DPBS.

In Vivo Biodistribution Studies:

Athymic nude mice (8 week, female) with a tumour volume of ˜300 mm³,were injected with 3.2 mg/ml (12.8 mg/kg) of TiO₂-AlexaTf (n=5) andAlexaTf (n=5) (Life Technologies Inc.) in DPBS (100 μl) intravenouslythrough the lateral tail vein. Fluorescence imaging was performed usingexcitation and emission wavelengths of 685 nm and 720 nm, respectively,in a Pearl whole animal imager (Li-Cor Biosciences Inc., Lincoln, USA).The mice were euthanized 24 h post-injection and the major organs weredissected and imaged. Mean fluorescence intensity for each tissue wasestimated by region of interest analysis using Pearlcam software (Li-CorBiosciences Inc.). The intensity was normalized to equalize musclefluorescence levels and plotted for all the organs. For TiO₂ alone, themice were injected with 250 μg/ml of TiO₂-PEG in DPBS (100 μl)intravenously through tail vein. The organs were dissected andfluorescence imaging was performed using Kodak IS4000MM multimodalimaging system (Carestream Health Inc., Rochester, USA) withexcitation/emission wavelength set to 640/700 nm, 60 s exposure with 2×2binning, for detecting TiO₂ using its inherent fluorescence.

In Vivo Blocking Studies:

In Athymic nu/nu mice (8 week, female) bearing HT1080 tumours, 200 mg/kgof holo-Tf (Sigma Aldrich Co.) was administered i.v. After 45 min,TiO₂-AlexaTf (10 mg/kg) was administered, and imaging was performed asdescribed above. The animals were euthanized 24 h post injection for exvivo biodistribution analysis of the organs.

CRIT of Solid Tumours:

For intratumoural administration, a cocktail of 2.5 μg/ml of TiO₂-PEGand 0.5 mCi/0.1 ml chelated ⁶⁴Cu in 50 μl of DPBS was injected directlyinto the tumour mass, after the tumour volume reached 200 mm³. Twodiametrically opposite injection sites were chosen and 25 μl of thecocktail was delivered at each site. Four groups (n=4), TiO₂-PEG treatedmice, ⁶⁴Cu treated mice, non-radioactive Cu (1 μM CuCl₂) treated miceand untreated mice, served as controls.

For systemic administration, when tumour volume reached 50 mm³, the mice(n=6 per group) were injected with 1 mg/kg TiO₂-Tf, Tc-Tf, TiO₂-Tf-Tc in100 μl of DPBS intravenously followed by 0.87 mCi/0.1 ml of FDG, alsointravenously, 24 h later. Control mice (n=6 per group) wereadministered with DPBS, the constructs or FDG alone. Animals wererandomly divided into three blocks of two animals each for differenttreatments. Food was withheld from mice for 6 h before administering FDGand kept in a dark, lead-shielded room post injection. A secondadministration of FDG (0.87 mCi/0.1 ml) was given 48 h after the firstFDG injection. Similarly, two additional cohorts were administered with0.14 mCi/0.1 ml and 0.43 mCi/0.1 ml FDG (n=4 per group), respectively,and monitored over 45 d. For mice undergoing treatment using external UVlight irradiation, the tumours were irradiated directly by a mercurylamp (300-400 nm) for I h at 14-20 J/cm², 24 h after administration ofTiO₂-Tf-Tc constructs. Irradiation was reapplied again after 48 h andthe cycle repeated 2×. For both, systemic and intratumoural studies, themice were monitored for 45 days and the growing tumours were measuredwith callipers every two days and tumour volume calculated using theequation: =(length×width²)/2. The tumour volume was plotted versus timeto analyse CRIT effect on the different groups of mice. The weight andany physical signs for distress were also monitored closely. The micewere euthanized by cervical dislocation after anaesthesia with 5%isoflurane when the tumour size reached 2 cm or loss of >20% total bodyweight. The mice with regressing tumours were monitored for anadditional four months to determine whether the cancer was in remission.Similarly, CRIT was performed on slow growing A549 xenograft models andtumor growth monitored for 35 days.

FDG-PET Imaging:

FDG-PET imaging was performed on untreated mice on day 15 and on treatedmice on day 30. The mice were fasted for 6 h before each scan. Afteranesthetizing the mice with 1.5-2% Isoflurane and Oxygen, 0.19 mCi (7.03MBq)/0.1 ml of FDG was administered through i.v. route. A ten-minutetransition scan was performed just before the ten minute emission at 1 hpost injection using a MicroPET-Inveon MultiModality scanner (SiemensPreclinical Solutions, Erlangen, Germany). The animals were placed onthe microCT® in the same position to obtain anatomical imaging andco-registered to the microPET® image. The data were analysed usingInveon Research Workstation software, by manually drawing 3-dimensionalregions of interest (ROI) from PET images using CT anatomicalguidelines. The activity associated with tumour was measured and maximumstandard uptake values (SUVs) were calculated usingSUV=([nCi/mL]×[animal weight (g)]/[injected dose (nCi)]).

Histology:

The HT1080 tumour bearing mice in the control groups were euthanized 15d post administration of TiO₂-Tf, Tc-Tf, TiO₂-Tf-Tc constructs or FDG,while mice treated with 1 mg/kg of TiO₂-Tf and Tc-Tf with FDG wereeuthanized 30 d post administration and the group treated with 1 mg/kgof TiO₂-Tf-Tc with FDG were euthanized at 45 d post administration.Similarly, mice treated by intratumoural administration of TiO₂-PEG and⁶⁴Cu were euthanized for histology 3 d after treatment. The tumours wereharvested and snap-frozen in Optimal Cutting Temperature (OCT) media forroutine staining with haematoxylin and eosin (H&E). Brightfield imagesof H&E stained 10 μm tumour sections were obtained byepifluorescencemicroscopy at 4× and 20× magnifications and analysed by a pathologist.

Statistical Analysis:

Statistical significance was measured by Student's t-test using GraphPad Prism software (Graph Pad, San Diego, Calif.). Kaplan-Meir survivalcurves were plotted using Graph Pad Prism software. Unless notedotherwise, all values are means and error bars are standard deviations.For animal studies, sample size estimates depend on the effect size(mean difference between untreated and treatment groups/SD) of outcome.For effect size of 2.1 and using two-sided t-test, typically 5 per groupwere needed with 80% power to detect a significant difference at a typeI error rate of 0.05.

Example 23 CRIT in Multiple Myeloma

Transferrin receptor (TfR) is over expressed on highly proliferatingcells, which includes most tumor types, due to the increased ironrequirement for DNA synthesis. Moreover, in multiple myeloma, ironmetabolism is significantly altered, which typically manifests as anemiain more than 73% of patients. Flow cytometry using anti-CD71 antibodieslabeled with phycoerythrin show that TfR expression was upregulated invarious MM cell lines, including 5TGM, U266, and MM1.S (>98%) comparedto T cells (2%) and B cells (25%) (FIG. 35). T cells were identifiedusing CD4 antibody and B cells using CD19 antibody. This demonstratesthat the unexplored Tf is a viable homing ligand to MM cells.

Having demonstrated the complementary effects of Tc and TiO₂nanoparticles in the HT1080 model, we applied the concept to MM. Cellviability studies of 5TGM and U266 using the MTS assay revealed thatwhen cells were treated with both 10 μg/mL of TiO₂-Tf-Tc and 31 MBq/0.1mL of ¹⁸FDG, significantly higher cell death occurred (FIG. 36). Minimalcell death was observed in untreated cells and the control groups.Staurosporine (2 μM) was used as a positive control.

Biodistribution studies were carried out in NSG mice with U266subcutaneous xenograft tumors using Alexa 680 labeled Tf conjugated toTiO₂. The fluorescence of Alexa 680 dye labeled Tf was used fornon-invasive determination of the in vivo distribution andtumor-selective uptake of the nanoparticle construct (FIG. 37). TiO₂-Tfuptake was highest in tumors relative to other organs, an outcome thatis rare for most nanoparticles. The high tumor-to-muscle ratio of 23.5and low uptake by liver, kidney, and spleen could be attributed toTf-mediated endocytosis. The Tf receptor has a fast turnover rate,enabling multiple cycles of nanoparticle endocytosis.

Example 24 CRIT Composition Comprising Micelles

Development and Characterization of Titanocene Loaded Lipid-MicellarNanoparticles:

For this project, the surfactant co-mixtures included 2 mole % of Tc,0.15 mole % of VLA-4-homing ligand conjugated lipids, and ˜96.5 mole %of lecithin (FIG. 38). Hydrodynamic particle size was 16±4 nm, with anarrow distribution (polydispersity indexes, PDI: ˜0.1-0.2). Thenegative electrophoretic potential (ca.-20±6 mV) point to the colloidalstability and successful lipid encapsulation. TEM images of micellesalone confirmed a spherical shape in the anhydrous state (FIG. 38B). Theelectron dense signatures in the periphery and increase in opacity inthe center compared to micelles alone demonstrates the successfulincorporation of Tc in the lipid membrane and the center of thenanomicelles (FIG. 38C). These particles possess long shelf-lifestability and retain the particle integrity (>5 months to date) over abroad pH range (pH 5.6-9.4). The UV-vis spectra of Tc (λ_(max) 250 nm;plus another 322 nm peak) show an excellent overlap with thepredominantly UV emission of CR for CRIT.

Demonstration of In Vitro CRIT in MM Cells:

To delineate intrinsic from CR-mediated toxicity, we carried outtoxicity analysis on micelles+Tc or ¹⁸FDG alone using 5TGM and U266 MMcell lines. A tetrazolium dye based MTS cell viability assay shows thatcells treated with 5 μg/mL of micelles+Tc or 31 MBq/0.1 mL of ¹⁸FDGwere >95% viable. When treated with ¹⁸FDG, the viability of MM cellspretreated with micelle+Tc significantly decreased (FIG. 39), suggestinglow metabolic activity and attenuated proliferation. We usedStaurosporine (2 μM) as a positive control.

Establishment of Animal Models and Development of Imaging Agents forNoninvasive Imaging and Monitoring of Treatment Response:

We have established both GFP and luciferase-expressing MM in KaLwRijmice (FIG. 40A,C). These models will be used to monitor treatmentresponse noninvasively and longitudinally. We have also demonstrated themetabolic and VLA-4 targeted PET imaging of MM in orthotopic mousemodels with ¹⁸FDG and ⁶⁴Cu-labeled VLA-4 ligand (FIG. 40). These datademonstrate the availability of realistic animal models andradiopharmaceuticals for imaging, treating, and monitoring treatmentresponse of MM to CRIT.

Pharmacokinetics and Biodistribution of Tc Loaded VLA-4 TargetedNanomicelles:

Pharmacokinetics of targeted micelle+Tc (25 μL/kg) was evaluated in ratsby inductively coupled plasma optical emission spectrometry (ICP OES)using the Ti signal from Tc. A half-life of 123 min was obtained (FIG.41A), which is consistent with previous data using similar nanomicellesloaded with gadolinium (122 min). ICP OES biodistribution analysis ofthe targeted micelle+Tc revealed significantly higher uptake andretention in MM than other organs 24 h post injection (FIG. 41B).

Demonstration of In Vivo CRIT Using 5TGM Xenograft MM Mouse Model:

5TGM subcutaneous xenograft tumors were grown in KaLwRij mice andtreatment was initiated once the tumors became palpable. An intravenous(i.v.) dose (50 μL) of micelle+Tc containing 0.3 mg/kg of Tc wasadministered, followed by an i.v. dose of 31 MBq/0.1 mL of ¹⁸FDG after 6h. The same dosing schedule was followed for non-targeted micelle+Tc andmicelles alone. Median survival increased from 10±2 d, for the untreatedand control groups, to 17±2 d for mice treated with targeted micelle+Tcwith ¹⁸FDG (FIG. 42A), a vast improvement for MM therapy. We evaluatedif the expression of GFP in 5TGM cells is altered as a result of cellsundergoing apoptosis in ex vivo tumor samples. The tumors that underwentCRIT with targeted micelle+Tc (FIG. 42C) had significantly lower GFPfluorescence compared to untreated tumors (FIG. 42B), suggestingperturbation in protein expression as a result of either free radicaldamage or a direct consequence of cells undergoing apoptosis.

1. A composition, the composition comprising at least onelight-sensitive molecule complexed with a targeting agent.
 2. Thecomposition of claim 1, further comprising a second light-sensitivemolecule.
 3. (canceled)
 4. The composition of claim 1, wherein the atlight sensitive molecule is selected from the group consisting ofphotosensitizers, photoinitiators, and photocatalysts.
 5. Thecomposition of claim 2, wherein the light-sensitive molecules are aphotocatalyst, and a photoinitiator.
 6. The composition of claim 1,wherein the light-sensitive molecule is TiO₂ nanoparticles.
 7. Thecomposition of claim 1, wherein the light-sensitive molecule istitanocene.
 8. The composition of claim 2, wherein the atlight-sensitive molecules are TiO₂ nanoparticles and titanocene. 9.(canceled)
 10. The composition of claim 2, wherein the targeting agentis transferrin and the light-sensitive molecules are titanocene and TiO₂nanoparticles.
 11. The composition of claim 1, wherein the targetingagent has affinity for a tumor.
 12. The composition of claim 1, whereinthe targeting agent is transferrin. 13.-15. (canceled)
 16. Thecomposition of claim 1, wherein the composition is activated byCR-emitting radionuclides.
 17. The composition of claim 16, wherein theCR-emitting radionuclides are selected from the group consisting ¹⁸F,¹⁸F-FDG, ⁶⁴Cu, ⁹⁰Y, ¹²⁴I and ⁸⁹Zr. 18.-22. (canceled)
 23. A method foradministering Cerenkov radiation-induced therapy (CRIT) to a targettissue in a subject, the method comprising: a. administering to thesubject an effective amount of a composition comprising at least oneradiation-sensitive molecule and a targeting agent; and b. administeringto the subject an amount of a Cerenkov radiation (CR)-emittingradionuclide effective to activate the radiation-sensitive molecule,thereby administering CRIT to the target tissue in the subject.
 24. Themethod of claim 23, wherein administering CRIT to a target tissue isused to treat a disease associated with the target tissue. 25.-26.(canceled)
 27. The method of claim 23, wherein the CR-emittingradionuclide is administered at a 2-fold lower dose than standardtreatment. 28.-31. (canceled)
 32. The method of claim 23, whereincomposition comprises two radiation-sensitive molecules.
 33. The methodof claim 23, wherein the radiation-sensitive molecule is selected fromthe group consisting of TiO₂ and titanocene. 34.-41. (canceled)
 42. Amethod for treating a tumor in a subject, the method comprising: a.administering to the subject an effective amount of a compositioncomprising at least one radiation-sensitive molecule and a targetingagent; and b. an amount of a Cerenkov radiation (CR)-emittingradionuclide effective to activate the radiation-sensitive molecule,thereby treating the tumor.
 43. (canceled)
 44. The method of claim 42,wherein composition comprises two radiation-sensitive molecules.
 45. Themethod of claim 42, wherein the radiation-sensitive molecule is selectedfrom the group consisting of TiO₂ and titanocene.